CN118416207B

CN118416207B - A paratuberculosis subunit vaccine and its preparation method and application

Info

Publication number: CN118416207B
Application number: CN202410875784.6A
Authority: CN
Inventors: 刘思国; 党光辉; 崔莹莹
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2024-07-02
Filing date: 2024-07-02
Publication date: 2025-02-11
Anticipated expiration: 2044-07-02
Also published as: CN118416207A

Abstract

The invention discloses a paratuberculosis subunit vaccine, a preparation method and application thereof. The vaccine contains 89AP recombinant antigen, wherein the 89AP recombinant antigen is obtained by fusion expression of MAP3527, MAP1609c and p22 genes of mycobacterium paratuberculosis (Mycobacterium avium subsp. Paratuberculosis, MAP). The research shows that the recombinant antigen 89AP can induce mice to generate strong cellular immunity and humoral immunity reaction, can provide good protection effect for MAP infection, and can not interfere quarantine of tuberculosis and paratuberculosis, so that 89AP is an ideal subunit candidate vaccine for paratuberculosis, and provides a new technical means for developing novel vaccines for paratuberculosis.

Description

Paratuberculosis subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to a paratuberculosis subunit vaccine, a preparation method and application thereof, and belongs to the technical field of medicines.

Background

Paratuberculosis (Paratuberculosis), also known as Johne's disease, JD, is a chronic wasting infectious disease caused by mycobacterium paratuberculosis (Mycobacterium avium subsp. Paratuberculosis, MAP) that causes granulomatous enteritis in ruminants such as cattle and sheep, and is a typical condition, JD clinically manifests as refractory diarrhea, decreased milk production, severe wasting, anemia and somnolence, eventually dying from exacerbation. JD not only causes a huge economic loss to the livestock breeding industry, but also threatens public health safety.

Because JD has the problems of long latency, insignificant subclinical symptoms, poor treatment effect, imperfect detection methods, etc., in order to reduce the risk of infection, the main prevention and control strategy for killing diseased animals is still to be adopted at present. However, detection and killing of positive animals has not achieved the expected effect so far, and thus it is necessary to pay attention again to JD vaccine, and immunoprophylaxis remains an effective way to prevent JD problems worldwide. The ideal vaccination not only can effectively reduce economic loss caused by killing and MAP dropping of diseased animals, but also can effectively reduce clinical infection cases and the like. At present, JD vaccines are mainly divided into three major classes, namely whole cell inactivated vaccines, attenuated live vaccines and subunit vaccines.

Currently, common commercial MAP whole cell inactivated vaccines include Mycopar, gudair and Silirum. Mycopar consists of inactivated Mycobacterium avium subspecies 18 strain (P18) and mineral oil adjuvant, can reduce clinical morbidity and MAP shedding after inoculation, is suitable for herds with high MAP infection rate or herds with limited implementation of paratuberculosis control measures, but the vaccine is derived from P18 strain of Mycobacterium avium subspecies (MAA), can not provide immunogenicity completely similar to MAP, has no effect on reducing the MAP quantity in infected tissues, and can also interfere with quarantine and JD diagnosis of bovine tuberculosis. The attenuated live vaccine is prepared from MAP attenuated strain and oil adjuvant, and is used for immunoprophylaxis of cattle and sheep JD. MAP attenuated strains are obtained by knocking out 1 or more virulence genes of MAP by adopting technologies such as transposon mutation, allele exchange, phage mediation and the like, and the MAP attenuated strains are proved to have good virulence reduction and immunogenicity. Studies show that the attenuated live vaccine can induce high-level IFN-gamma production and has obvious protection effects in the aspects of reducing inflammation, eliminating acid-fast bacteria and the like. Attenuated live vaccines employing a variety of antigen libraries are capable of eliciting immunoprotection against MAP infection by stimulating innate and adaptive immunity. After inoculation of MAP transposon mutants, the spleen bacterial load of mice was significantly reduced. In a small ruminant infection model, two LAV candidate deletion strains MAP delta relA and MAP delta leuD obtained by using an allele exchange technology have a protection effect on the challenge of immune goats and calves, and can reduce the shedding of MAP in feces, but 80% of animals still have lesions in an infection experiment in immune sheep, and the cattle can interfere with quarantine of bovine tuberculosis after being vaccinated with MAP attenuated live vaccines, and risk of virulence return exists, especially in individuals with low immune functions and calves. Subunit vaccine is prepared from recombinant MAP protein with definite components or DNA encoding immunogenic antigen, and compared with attenuated live vaccine, the subunit vaccine has no toxicity, no risk of strong return, reduced inflammation and granuloma formation at inoculation site, and no interference to bovine tuberculosis and JD quarantine. However, none of the subunit vaccines tested to date provide complete protection in the mouse infection model. Subunit vaccines have a short half-life in vivo and therefore often require multiple immunizations and the use of adjuvants to achieve protective effects.

MAP3527 is a trypsin-like serine protease containing a PDZ domain at the C-terminus. MAP3527 and Rv0125 have homologous regions, the amino acid homologous regions are up to 75.4%, and Rv0125 can induce CD4+ and CD8+ T cells to generate IFN-gamma, which indicates that Rv0125 is a good protein antigen. P22 is an immunogenic MAP lipoprotein, belonging to the Mycobacterium lipoprotein LppX/LprAFG family. P22 is also a candidate protein for serological detection of MAP, and antibodies against P22 protein can be detected in naturally infected cattle. Ag85B (MAP 1609 c) belongs to one of the Ag85 protein family members, which is the most secreted group of proteins with phytase activity in mycobacteria. It is reported that recombinant protein 66NC constructed by 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 a good protection effect on MAP attack.

In summary, since there is a defect in the existing paratuberculosis vaccine due to the lack of effective treatment methods and the increase of raising cost caused by the elimination of diseased animals, the development of a novel paratuberculosis vaccine with good immune effect has become an urgent need for immunoprophylaxis.

Disclosure of Invention

The invention aims to provide a paratuberculosis subunit vaccine, and a preparation method and application thereof.

In order to achieve the above purpose, the invention adopts the following technical means:

The invention constructs 2 recombinant plasmids pET-28a-3527N-1609C-P22-3527C and pET-28a-3527N-P22-1609C-3527C by utilizing MAP3527, MAP1609C (Ag 85B) and P22 genes of MAP, transfers the recombinant plasmids into escherichia coli BL21 for culturing and expressing, successfully obtains 2 recombinant proteins after purification by Ni column affinity chromatography, and is respectively named as 89AP and 89PA according to the size and arrangement mode. Using purified 2 recombinant proteins 89AP and 89PA, the MONTANIDE ISA 61 VG adjuvant was emulsified with the protein, injected subcutaneously in multiple spots, and immunized mice were screened twice every three weeks with IFN- γelispot experiments, compared to the reported Ag 85B-containing fusion protein 66NC, P22-containing fusion protein 58F, and the ag85b+p22 mixed protein, and PBS was used as a negative control. The results show that the recombinant antigen 89AP can induce mice to produce high levels of IFN-gamma, which is obviously higher than control proteins (66 NC, 58F, 66NC+58F and Ag85B+P22 mixed proteins), and the immune effect of the 89AP is better than that of the control proteins.

In addition, in order to verify the intradermal allergic reaction condition of the bovine tuberculin and the paratuberculin after 89AP protein immunization of guinea pigs, the invention applies the bovine tuberculin and the paratuberculin to carry out intradermal allergic reaction detection on the 89AP protein and other immunized guinea pigs, and compares the allergic reaction difference of the 89AP immune group and 66NC immune group, BCG immune group, K-10 immune group and PBS control group. The results show that under the condition that the BCG immunity, the K-10 immunity positive control result and the PBS negative control result are all established, no delayed type allergy appears at the inoculation positions of the 89AP immunity group and the 66NC immunity group (48 h), and strong allergic reactions appear on the 89AP protein stimulation and the 66NC immunity group on the 66NC protein stimulation, so that the 89AP protein does not interfere with quarantine of tuberculosis and paratuberculosis after the 89AP protein is used for immunizing guinea pigs, therefore, the 89AP is an ideal candidate recombinant subunit vaccine and can be used as a candidate subunit vaccine for preventing JD.

Based on the research, the invention provides a paratuberculosis subunit vaccine, which contains 89AP recombinant antigen, wherein the 89AP recombinant antigen is obtained by fusion expression of the genes of map3527, map1609c and p22 of paratuberculosis mycobacterium, and the amino acid sequence of the 89AP recombinant antigen is shown as SEQ ID NO.1.

Wherein, preferably, the 89AP recombinant antigen is obtained by a prokaryotic expression system.

Preferably, the 89AP recombinant antigen is obtained by transferring a recombinant plasmid pET-28a-3527N-1609C-P22-3527C into escherichia coli BL21 for culture and expression, and purifying by Ni column affinity chromatography, wherein the recombinant plasmid pET-28a-3527N-1609C-P22-3527C is constructed by the following method:

(1) PCR amplification of the p22 Gene:

amplifying p22 gene with primer p22-F/R by using extracted MAP K-10 strain whole genome DNA as template, and freeze preserving at-20 deg.C with gel recovering kit after the PCR product is identified correctly;

;

(2) Single cleavage of pET-28a-3527N-1609C-3527C vector:

Single enzyme cutting pET-28a-3527N-1609C-3527C vector with HindIII, recovering with gel recovering kit after enzyme cutting, and freeze preserving at-20deg.C;

(3) Ligation of the cleavage vector with PCR product:

respectively connecting the pET-28a-3527N-1609C-3527C digestion products with P22 products to construct recombinant plasmids pET-28a-3527N-1609C-P22-3527C;

(4) Conversion of ligation products:

adding the connected products into E.coil DH5 alpha competent cells respectively, uniformly mixing, carrying out ice bath 30 min, immediately carrying out heat shock on the E.coil DH5 alpha competent cells in a 42 ℃ water bath for 30-45 s, immediately carrying out ice bath 2min again, adding 500 mu L of LB liquid medium, recovering the strain at 37 ℃ 180 rpm for 60 min, centrifuging at 3 rpm for 5 min, discarding the supernatant, re-suspending the precipitated thalli by using 100 mu L of LB liquid medium, coating the suspension on LB solid flat-plate medium containing 50 mu g/mL kanamycin, culturing at 37 ℃ in a incubator for 24 h to obtain single colonies, picking single colonies with good growth state, inoculating the single colonies into LB liquid medium containing 50 mu g/mL kanamycin at 5mL, culturing at 37 ℃ 180 rpm overnight, centrifuging the bacterial liquid at 3 rpm for 5 min, extracting plasmids by using a plasmid small extraction kit, and carrying out PCR and sequencing identification on recombinant plasmids pET-28a-3527N-1609C-P22-3527C;

(5) Induction expression of 89AP protein:

(5.1) construction of the over-expressed protein BL21 strain:

Transferring 5-10 mu L of recombinant plasmid pET-28a-3527N-1609C-P22-3527C identified correctly into 50 mu L of E.coil BL21 competent cells;

(5.2) inducible expression of recombinant proteins:

BL21 single colonies were picked up in LB solid medium, inoculated in 5mL LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃180: 180 rpm for 24 h. Transferring 100 mu L of bacterial liquid into 10mL LB liquid culture medium containing 50 mu g/mL kanamycin, continuously culturing until OD ₆₀₀ is approximately equal to 0.8, collecting 1 mL whole bacterial liquid sample preparation and preservation before induction, adding IPTG with final concentration of 1mM into the rest bacterial liquid, inducing protein expression 4 h at 37 ℃ 180 rpm, and collecting 1 mL whole bacterial liquid sample preparation and preservation after induction;

(5.3) identification of recombinant proteins:

Western blot is used for analyzing the induction expression condition of the recombinant protein, and the recombinant protein is named 89AP;

(6) 89AP protein purification.

Preferably, the step (6) includes the following steps:

(6.1) picking up the identified BL21 single colony from LB solid plate medium, inoculating to LB liquid medium containing 50 mug/mL kanamycin in 10 mL, culturing 24h in 37 ℃ 180 rpm, transferring 10 mL bacterial liquid to LB liquid medium containing 50 mug/mL kanamycin in 1L, continuously culturing until OD ₆₀₀ is about 0.8, adding IPTG with final concentration of 1mM, inducing protein expression in 37 ℃ 180 rpm for 4h, centrifuging 10min at 4 ℃ 8 rpm, collecting bacterial precipitate, re-suspending each 1L bacterial precipitate by using 40mL Buffer A, and performing ice bath ultrasonic disruption, wherein the ultrasonic program is that the amplitude is 38%, ultrasonic 3 s, stopping 3 s and effective ultrasonic time is 30 min. Performing preliminary centrifugation at 4 ℃ and 3,500 rpm for 10min after ultrasonic crushing, removing precipitated fragments after centrifugation, and taking the supernatant and centrifuging at 10 rpm for 30, 30min again;

Wherein, buffer A is 20 mM Tris-HCl containing 150 mM NaCl and 10% v/v glycerol, and the pH value is 8.0;

(6.2) protein was expressed as inclusion bodies, the pellet after centrifugation was collected, resuspended in 80 mL Buffer B, stirred overnight at room temperature using a rotor until the solution was clear, centrifuged at 10 rpm at 4 ℃ for 30min, the supernatant was collected, diluted to a concentration of less than 1 mg/mL with Buffer B, transferred to dialysis bags, the supernatant in each 100 mL dialysis bag was soaked with 1L renaturation solution, after stirring at 4 ℃ for 18 h, the dialysis bag was replaced in Buffer a, and stirred at 4 ℃ for 18 h. Taking out supernatant in the dialysis bag after renaturation, centrifuging at 4 ℃ and 12 rpm for 30min, and collecting the supernatant;

wherein, buffer B is 20 mM Tris-HCl containing 150 mM NaCl,0.8% w/v SKL and 10% v/v glycerol, and the pH is 8.0;

The renaturation solution is 150 mM Tris-HCl containing 0.54 g reduced glutathione, 0.06 g oxidized glutathione, 150 mM NaCl and 3% v/v glycerol, and the pH value is 7.5;

(6.3) taking 20 mu L of supernatant sample before column hanging, and taking the supernatant sample as penetrating liquid;

(6.4) passing through the balance column material of the Ni-NAT chromatographic column by using 20 mL Buffer A, passing the supernatant through the chromatographic column, collecting the passing liquid, and repeatedly hanging the column for 6 times.

(6.5) Taking 20 mu L of supernatant sample after column hanging, and preparing a penetrating fluid;

(6.6) using 20 mL Buffer A flow through Ni-NAT chromatographic column to balance column material, adding 5 mL Buffer A heavy suspension column material, taking 20 mu L column material suspension, and taking out sample, and making resin pass through;

(6.7) passing through the columns by 40 mL Buffer C,40 mL Buffer D,40 mL Buffer E,40 mL Buffer F,20 mL Buffer G,20 mL Buffer H streams respectively to wash off the impurity proteins;

Wherein Buffer C is 20mM Tris-HCl containing 500 mM NaCl,3% v/v glycerol and 20mM imidazole, and has a pH of 8.0, buffer D is 20mM Tris-HCl containing 500 mM NaCl,3% v/v glycerol and 40 mM imidazole, has a pH of 8.0, buffer E is 20mM Tris-HCl containing 500 mM NaCl,3% v/v glycerol and 60mM imidazole, has a pH of 8.0, buffer F is 20mM Tris-HCl containing 500 mM NaCl,3% v/v glycerol and 80 mM imidazole, has a pH of 8.0, buffer G is 20mM Tris-HCl containing 500 mM NaCl,3% v/v glycerol and 80 mM imidazole, and has a pH of 8.0, and Buffer H is 20mM Tris-HCl containing 1M NaCl,3% v/v glycerol and 80 mM imidazole;

(6.8) using 20 mL Buffer A flow through Ni-NAT chromatographic column balance column material, adding 5 mL Buffer A heavy suspension column material, taking 20 mul L column material suspension, reserving sample, and making resin after impurity washing;

(6.9) after the column material is naturally precipitated, avoiding suspending the column material, slowly adding 5 mL Buffer I, standing on ice for 20: 20min, collecting naturally left eluent 1, adding 5 mL Buffer J, standing on ice for 20: 20min, collecting eluent 2, finally adding 5 mL Buffer K, standing on ice for 20: 20min, collecting eluent 3, and respectively taking 20 mu L of eluent as samples;

Wherein Buffer I is 20 mM Tris-HCl containing 150mM NaCl,5% v/v glycerol and 500 mM% imidazole, and has a pH of 8.0, buffer J is 20 mM Tris-HCl containing 150mM NaCl,5% v/v glycerol and 1M imidazole, and has a pH of 8.0, and Buffer K is 20 mM Tris-HCl containing 150mM NaCl,5% v/v glycerol and 2M imidazole, and has a pH of 8.0;

(6.10) using 20 mL Buffer A flow through Ni-NAT chromatographic column balance column material, adding 5 mL Buffer A heavy suspension column material, taking 20 mu L column material suspension, retaining sample, and making resin after elution;

(6.11) SDS-PAGE gel electrophoresis to identify the collected protein samples;

(6.12) according to the analysis result of SDS-PAGE gel electrophoresis, collecting protein eluent, using 30 kDa ultrafiltration concentration centrifuge tube, ultrafiltration desalting at 4 ℃ and 3 rpm, concentrating to volume below 1 mL, filtering and sterilizing with 0.45 μm filter, measuring protein concentration, and preserving at-80 ℃ for standby.

Wherein, preferably, the vaccine also comprises an adjuvant.

Wherein, preferably, the adjuvant is MONTANIDE ISA 61 VG adjuvant.

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

Furthermore, the invention also provides application of the paratuberculosis subunit vaccine in preparing medicines for preventing paratuberculosis.

Compared with the prior art, the invention has the beneficial effects that:

At present, development and application of recombinant protein subunit vaccines are important means for preventing infectious diseases, and a recombinant protein expression system and an advanced purification technology provide a new solution for novel vaccine development. The recombinant protein can improve the immunogenicity, protein half-life, protein solubility, etc. of the target protein by using a gene fusion technique. Recombinant protein expression systems include bacterial, yeast, baculovirus, mammalian cells, cell-free systems, and the like, and can induce a strong cellular immune response by means of antigens expressed by plasmids, bacterial or viral vectors. The escherichia coli has the advantages of easiness in operation, low cost, high expression speed, high yield and the like, and is the preferred host for the expression of the recombinant protein at present. According to MAP3527, p22 and MAP1609c genes, 2 recombinant proteins 89AP and 89PA taking inclusion bodies as expression modes are obtained through recombinant plasmids constructed by escherichia coli expression, correctly folded recombinant expression proteins are obtained through ultrasonic cleavage, protein denaturation and renaturation treatment, and 2 high-purity recombinant proteins are obtained after ultrafiltration and desalination and concentration after Ni column affinity chromatography purification.

The invention expresses and purifies 2 recombinant proteins 89AP and 89PA based on P22, MAP1609c and MAP3527c protein coding genes, so as to screen MAP subunit vaccine candidate recombinant antigens with good immunogenicity and protection effect. The prepared recombinant proteins 89AP, 89PA, 66NC, ag85B+P22 and 66NC+58F are emulsified with MONTANIDE ISA 61 VG adjuvant to immunize mice, and the screened MAP subunit vaccine candidate recombinant antigen 89AP is distinguished by an IFN-gamma ELISPOT method, so that the mice can be induced to generate strong cellular immunity and humoral immunity reaction, good protection effect can be provided for MAP infection, tuberculosis and quarantine of paratuberculosis can not be interfered, and 89AP is an ideal subunit candidate vaccine for paratuberculosis, and an important scientific basis is laid for research and development of novel paratuberculosis vaccines.

Drawings

FIG. 1 is a schematic diagram of recombinant plasmids;

wherein a is a pET-28a-3527N-1609C-P22-3527C recombinant plasmid pattern diagram, and b is a pET-28a-3527N-P22-1609C-3527C recombinant plasmid pattern diagram;

FIG. 2 shows agarose gel electrophoresis of PCR amplified products of the p22 gene and map1609c gene;

Wherein a is agarose gel electrophoresis diagram of a p22 gene PCR amplified product, lane M is DNA standard molecular weight DL2000, lane 1 is p22 gene PCR product, b is agarose gel electrophoresis diagram of a map1609c gene PCR amplified product, lane M is DNA standard molecular weight DL2000, lane 1 is map1609c gene PCR product;

FIG. 3 is a diagram of PCR identification of pET-28a-3527N-1609C-P22-3527C and pET-28a-3527N-P22-1609C-3527C recombinant plasmids;

Wherein a is a PCR identification chart of pET-28a-3527N-1609C-P22-3527C recombinant plasmid, lane M is DNA standard molecular weight DL2000, lane 1 is P22 gene PCR product, b is a PCR identification chart of pET-28a-3527N-P22-1609C-3527C recombinant plasmid, lane M is DNA standard molecular weight DL2000, lane 1 is map1609C gene PCR product;

FIG. 4 is a Western blot analysis protein induction expression pattern;

Wherein, lane M is protein Marker, lane 1 is 89AP_BL21 pre-induction whole bacteria, lane 2 is 89AP_BL21 post-induction whole bacteria, lane 3 is 89AP_BL21 post-ultrasound supernatant, lane 4 is 89AP_BL21 post-ultrasound precipitation, lane 5 is 89PA_BL21 pre-induction whole bacteria, lane 6 is 89PA_BL21 post-induction whole bacteria, lane 7 is 89PA_BL21 post-ultrasound supernatant, lane 8 is 89PA_BL21 post-ultrasound precipitation;

FIG. 5 is a SDS-PAGE detection protein purification chart;

wherein a is an SDS-PAGE identification 89AP purification chart, lane M is a protein Marker, lane 1 is a pre-threading liquid, lane 2 is a threading liquid, lane 3 is a threading resin, lane 4 is a post-hybridization resin, lane 5:500mM imidazole eluate 1, lane 6:1M imidazole eluate 2, lane 7:2M imidazole eluate 3, lane 8 is a post-elution resin, b is an SDS-PAGE identification 89PA purification chart, lane M is a protein Marker, lane 1 is a pre-threading liquid, lane 2 is a threading liquid, lane 3 is a threading resin, lane 4 is a post-hybridization resin, lane 5:500mM imidazole eluate 1, lane 6:1M imidazole eluate 2, lane 7 is a post-elution resin;

FIG. 6 is a SDS-PAGE detection of 66NC protein purification;

Wherein, lane M is protein Marker, lane 1 is penetrating fluid, lane 2 is penetrating fluid, lane 3 is penetrating resin, lane 4 is post-eluting resin, lane 5 is eluting with 500mM imidazole 1, lane 6 is eluting with 1M imidazole 2, lane 7 is eluting with 2M imidazole 3, and lane 8 is post-eluting resin;

FIG. 7 is a SDS-PAGE detection 58F protein purification;

Wherein, lane M is protein Marker, lane 1 is penetrating fluid, lane 2 is penetrating fluid, lane 3 is penetrating resin, lane 4 is resin after washing, lane 5 is eluting with 60mM imidazole, lane 6 is eluting with 80mM imidazole, lane 7 is eluting with 100mM imidazole, lane 8 is eluting with 200mM imidazole, lane 9 is eluting with 500mM imidazole, and lane 10 is resin after eluting;

FIG. 8 is a graph showing the results of detection of IFN-gamma expression in spleen lymphocytes of immunized mice by an ELISA spot analyzer;

Wherein a is a chart for observing IFN-gamma formation condition by an ELISA analyzer, and b is a statistical chart for IFN-gamma spot number;

FIG. 9 is a flow chart of mouse immunization;

FIG. 10 is a graph showing the results of detection of mouse serum-specific antibodies;

wherein a is an IgG antibody detection result diagram, b is an IgM antibody detection result diagram;

FIG. 11 is a diagram showing IFN-gamma expression after detection of 3W by ELISA;

Wherein a is a result chart of the formation condition of IFN-gamma observed by an enzyme-linked spot analyzer, and b is a statistical chart of the quantity of IFN-gamma spots;

FIG. 12 shows IL-4 expression after detection of 3W by ELISA;

Wherein a is a result graph of the formation condition of IL-4 observed by an ELISA (enzyme-Linked immunosorbent assay) analyzer, and b is a statistical graph of the number of IL-4 spots;

FIG. 13 shows IL-17A and IFN-gamma levels in mouse serum detected by ELISA at a level of 3W of the second boost;

FIG. 14 is a graph showing the results of ICS assay of the percent cytokine positive (% Cyt+) antigen-specific T lymphocyte reaction following a 3W-fold boost;

Wherein, a is a cytokine positive percentage (% Cyt+) antigen-specific CD ⁴⁺ T lymphocyte reaction result graph, and b is a cytokine positive percentage (% Cyt+) antigen-specific CD ⁸⁺ T lymphocyte reaction result graph;

FIG. 15 is a graph showing the results of serum cytokine detection in mice after MAP infection 2W;

FIG. 16 is a graph showing the results of ICS assay of the percent cytokine positive (% Cyt+) antigen-specific T lymphocyte reaction after MAP infection 2W;

Wherein, a is a cytokine positive percentage (% Cyt+) antigen-specific CD4+ T lymphocyte reaction result graph, and b is a cytokine positive percentage (% Cyt+) antigen-specific CD8+ T lymphocyte reaction result graph;

FIG. 17 is a graph showing the results of pathological organ injury in mice after MAP infection 2W;

Wherein a is a result graph of pathological damage of liver and intestinal tract after MAP infection 2W, and b is a result graph of pathological scoring of liver and intestinal tract after MAP infection 2W;

FIG. 18 is a graph showing the results of organ histopathological lesions of mice after MAP infection 2W;

Wherein a is a result graph of liver and intestinal tract histopathological damage after MAP infection of 2W, b is a result of liver histopathological scoring after MAP infection of 2W, c is a result graph of intestinal tract histopathological scoring after MAP infection of 2W;

FIG. 19 is a graph showing MAP loading and acid fast staining results in mouse tissues after MAP infection of mice;

wherein a is a colony colonization result graph of MAP in the liver of the mouse, b is a colony colonization result of MAP in the small intestine of the mouse, c is an acid-fast staining result graph after MAP infection by 2W;

FIG. 20 is a graph showing the results of guinea pig serum-specific antibody detection;

FIG. 21 is a graph showing the results of MAP guinea pig allergy detection.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The test methods used in the examples described below are conventional methods, and the materials, reagents, etc. used are commercially available ones, unless otherwise specified.

EXAMPLE 1 expression and immune evaluation of candidate recombinant antigens for paratuberculosis subunit vaccine

1. Experimental materials

1.1 Vectors and strains

Competent cells E.coli DH 5. Alpha. And E.coli BL21 were purchased from Tiangen Biological Company, pET28a vector, pET-28a-3527N-1609C-3527C (：Shao M, Cui N, Tang Y, 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（, which is described in the following: chen Fanre, zhang Jiajun, lemna minor, et al screening for Mycobacterium paratuberculosis immunogenic proteins and evaluation of immunoprotection effects [ J ]. Chinese agricultural science, 2024,57 (06): 1204-1214.) and MAP K-10 genomic DNA were stored and supplied by the laboratory.

1.2 Experimental animal

18C 57BL/6 female mice were purchased from Experimental animal technology Co., ltd, and fed to the Harbin veterinary institute of Endoctoral, china academy of agricultural sciences, and later study was conducted with the approval of the animal ethics committee (HVRI-IACUC-221108-02-GR).

1.3 Main reagent

2. Experimental method

2.1 Primer design and Synthesis

P22 and MAP1609c specific primers were designed using HindIII single cleavage according to the MAP K-10 reference strain genome sequence (NC_ 002944.2) published in GenBank. Primers were synthesized by the company Rayleigh Boxing, inc., see Table 2.

2.2 Construction of recombinant plasmids

2.2.1 Cultivation of MAP K-10 Strain

The MAP K-10 strain glycerinum preserved in a refrigerator at the temperature of minus 80 ℃ in a laboratory is inoculated into a mycobacteria ready-to-use liquid complete medium (containing 1 mg/L of mycobacteriin), placed in a shaking table at the temperature of 37 ℃ and 100 rpm for resuscitating and culturing until the OD600 reaches between 1.5 and 2.0, and the volume ratio of 1:100 is transferred into the mycobacteria ready-to-use liquid complete medium (containing 1 mg/L of mycobacteriin) and placed in a shaking table at the temperature of 37 ℃ and 100 rpm for culturing 2W.

2.2.2 Extraction of genome-wide DNA of MAP K-10 Strain

Taking 1 mL MAP K-10 bacterial liquid in a centrifuge tube, centrifuging at 3 rpm for 5 min, discarding supernatant, re-suspending bacterial precipitate with 1 mL of 20 mg/mL lysozyme, performing ice bath ultrasonic disruption by using an ultrasonic cell disruption instrument after acting on the bacterial precipitate in a 37 ℃ water bath for 2h, setting the amplitude to 20%, performing ultrasonic treatment for 3 s to 3 s, performing effective ultrasonic treatment for 3 min, and extracting MAP K-10 bacterial strain genome DNA by using a bacterial genome DNA extraction kit after ultrasonic treatment.

2.2.3 PCR amplification of p22 and map1609c genes

Using the extracted whole genome DNA of MAP K-10 strain as a template, the p22 and MAP1609c genes were amplified with p22-F/R and MAP1609c-F/R, respectively. The PCR products obtained were correctly identified, recovered using a gel recovery kit and the concentration was determined and stored frozen at-20 ℃.

2.2.4 Single cleavage of pET-28a-3527N-1609C-3527C and pET-28a-3527N-P22-3527C vectors

The pET-28a-3527N-1609C-3527C and pET-28a-3527N-P22-3527C vectors were cut singly using HindIII, the reaction system was 50. Mu.L, and after mixing 50. Mu.L of the above reaction system, incubated in a 37℃water bath for 30 min. And (3) after the enzyme digestion, the products are correctly identified, recovered by using a gel recovery kit, and the concentration is measured, and the products are frozen and stored in a refrigerator at the temperature of-20 ℃.

2.2.5 Ligation of the cleavage vector with the PCR product

The cleavage products of pET-28a-3527N-1609C-3527C and pET-28a-3527N-P22-3527C were ligated with the P22 and map1609C products, respectively, to construct two recombinant plasmids as shown in FIG. 1. And (3) uniformly mixing the reaction systems, and then incubating the mixture in a water bath at 50 ℃ for 15 min to obtain a connected product.

2.2.6 Conversion of ligation products

2 Kinds of connected products are added into E.coil DH5 alpha competent cells respectively, the mixture is evenly mixed, then is subjected to ice bath 30min, and is immediately subjected to heat shock 30-45 s in a 42 ℃ water bath, and then is immediately subjected to ice bath 2 min. 500. Mu.L of LB liquid medium was added, resuscitated at 37℃180 rpm for 60 min, centrifuged at 3 rpm for 5min, the supernatant was discarded, and the precipitated cells were resuspended in 100. Mu.L of LB liquid medium, spread on LB solid plate medium containing 50. Mu.g/mL kanamycin, and incubated at 37℃in an incubator for 24 h to obtain single colonies.

2.2.7 Identification of recombinant plasmids

Single colonies with good growth state are selected from LB solid medium, inoculated into 5mL LB liquid medium containing 50 mug/mL kanamycin, cultured overnight at 37 ℃, bacterial liquid is centrifuged at 3 rpm for 5min, plasmids are extracted by using a plasmid small extraction kit, recombinant plasmids pET-28a-3527N-1609C-P22-3527C and pET-28a-3527N-P22-1609C-3527C are identified by referring to a 2.2.3 PCR method, and plasmids with specific destination bands are sent for sequencing for further verification.

2.3 Inducible expression of 89AP and 89PA proteins

2.3.1 Construction of the overexpression protein BL21 Strain

5-10. Mu.L of the recombinant plasmids pET-28a-3527N-1609C-P22-3527C and pET-28a-3527N-P22-1609C-3527C, respectively, were transferred into 50. Mu.L of E.coil BL21 competent cells, transformation method referenced 2.2.6.

2.3.2 Inducible expression of recombinant proteins

BL21 single colonies were picked up in LB solid medium, inoculated in 5 mL LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃180: 180 rpm for 24 h. Transferring 100 mu L of bacterial liquid into 10 mL LB liquid culture medium containing 50 mu g/mL kanamycin, continuously culturing until OD600 apprxeq 0.8, collecting 1:1 mL whole bacterial liquid sample preparation and preservation before induction, adding IPTG with final concentration of 1: 1mM into the rest bacterial liquid, inducing protein expression 4:4 h at 37 ℃ 180: 180 rpm, and collecting 1:1 mL whole bacterial liquid sample preparation and preservation after induction.

2.3.3 Identification of recombinant proteins

The induced bacterial liquid was centrifuged at 3.500 rpm for 10 min, the supernatant was discarded, bacterial pellet was resuspended in 1mL Buffer A (150 mM NaCl,10% glycerol, 20 mM Tris-HCl, pH 8.0), sonicated in an ice bath with an ultrasound program of 38% amplitude, 3 s, stopped 3 s, effective ultrasound time 2 min. The post-sonication bacterial liquid was centrifuged at 3.500 rpm for 10 min, and the post-sonication supernatant and pellet were collected and sampled separately, and the pellet samples were resuspended using 1.1 mL Buffer A.

Four prepared samples of the collected whole bacteria before induction, whole bacteria after induction, supernatant after sonication and precipitation after sonication were analyzed by SDS-PAGE gel electrophoresis. The induction expression of 2 recombinant proteins was analyzed by Western blot using Anit-His Tag antibody (1:1,000) for the primary antibody and Goat anti mouse IgG antibody (1:10,000), and the 2 recombinant proteins were designated 89AP and 89PA, respectively, according to the sequence of insertion of the recombinant plasmid and the size of the protein.

2.4 Purification of 89AP and 89PA proteins

(1) The identified BL21 single colony was picked from LB solid plate medium, inoculated into 10 mL LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃180: 180 rpm for 24 h. Transferring 10 mL bacterial liquid into 1L LB liquid medium containing 50 mug/mL kanamycin, continuously culturing until OD600 is approximately equal to 0.8, adding IPTG with a final concentration of 1mM, inducing protein expression for 4h at 37 ℃ 180 rpm, centrifuging at 4 ℃ 8 rpm for 10 min, collecting bacterial precipitate, and carrying out ice bath ultrasonic crushing after re-suspending each 1L bacterial precipitate by using 40 mL Buffer A, wherein the ultrasonic program is that the amplitude is 38%, ultrasonic wave is 3 s, stop is 3 s, and the effective ultrasonic time is 30 min. After ultrasonication, the pellet was centrifuged at 4℃and 3,500 rpm for 10 min, and the pellet was discarded, and the supernatant was centrifuged again at 10,000 rpm for 30, 30 min.

(2) The 2 proteins were all expressed as inclusion bodies, the pellet after centrifugation was collected, resuspended in 80mL Buffer B (150 mM NaCl,0.8% SKL,10% glycerol, 20 mM Tris-HCl, pH 8.0), stirred overnight at room temperature using a rotor until the solution was clear, centrifuged at 10 rpm at 4℃for 30min, the supernatant was collected, diluted to a concentration below 1 mg/mL with Buffer B, transferred to dialysis bags, the supernatant in each 100mL dialysis bag was soaked with 1L renaturation 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℃for 18 h, the dialysis bag was replaced in Buffer A, and stirred at 4℃for 18 h. After the completion of the renaturation, the supernatant was taken out of the dialysis bag, centrifuged at 12 rpm at 4℃for 30min, and the supernatant was collected.

(3) Taking 20 mu L of supernatant sample before column hanging, and preparing into pre-threading liquid.

(4) After passing through the Ni-NAT chromatographic column with 20 mL Buffer A flow-through balance column material, passing the supernatant through the chromatographic column, collecting the flow-through liquid, and repeatedly hanging the column for 6 times.

(5) Taking 20 mu L of supernatant sample after column hanging, and preparing the supernatant sample as penetrating liquid.

(6) The column material is balanced by using 20 mL Buffer A flow through Ni-NAT chromatographic column, 5 mL Buffer A weight of suspension column material is added, 20 mu L of column material suspension is taken for sample retention, and resin is used for passing through.

(7) The column was run with 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), 20mL Buffer G (500 mM NaCl,3% glycerol, 20 mM Tris-HCl, pH 8.0), 20mL Buffer H (1M NaCl,3% glycerol, 20 mM Tris-HCl,80 mM imidazole, pH 8.0), respectively, to wash out the contaminating proteins.

(8) And (3) passing through a Ni-NAT chromatographic column by 20 mL Buffer A flows, balancing the column, adding 5 mL Buffer A weight of suspension column, taking 20 mu L of column suspension, reserving a sample, and performing impurity washing on the resin.

(9) After the column had precipitated naturally, the column was kept from being suspended, 5mL Buffer I (150 mM NaCl,5% glycerol, 20 mM Tris-HCl,500 mM imidazole, pH 8.0) was slowly added, the naturally remaining eluent 1 was collected after 20min on ice, 5mL Buffer J (150 mM NaCl,5% glycerol, 20 mM Tris-HCl, 1M imidazole, pH 8.0) was then added, 20min on ice and eluent 2 was collected after 20min on ice, and finally 5mL Buffer K (150 mM NaCl,5% glycerol, 20 mM Tris-HCl, 2M imidazole, pH 8.0) was added, and eluent 3 was collected after 20min on ice. 20. Mu.L of each of the 3 eluates was sampled.

(10) And (3) passing through a Ni-NAT chromatographic column by 20 mL Buffer A flows, balancing the column, adding 5 mL Buffer A weight of suspension column, taking 20 mu L of column suspension, reserving a sample, and preparing the resin after elution.

(11) SDS-PAGE gel electrophoresis identifies the collected protein samples.

(12) According to the analysis result of SDS-PAGE gel electrophoresis, protein eluent is collected, and after ultrafiltration and desalination by using a 30 kDa ultrafiltration concentration centrifuge tube at 4 ℃ and 3 rpm, the concentration volume is below 1 mL. The protein concentration was determined by filtration sterilization with a 0.45 μm filter and stored at-80℃until needed.

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

66NC/58F refer to Shao Mingzhu, chen Fanre （Shao M, Cui N, Tang Y, 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; Chen Fanre, zhang Jiajun, duckweed, etc. screening of Mycobacterium paratuberculosis immunogen protein and evaluation of immunoprotection effect [ J ]. Chinese agricultural science, 2024,57 (06): 1204-1214.) the study report that pET-28a-3527N-Ag85B-3527C (66 NC) and pET-28a-3527N-P22-3527C (58F) were respectively transferred into E.coli BL21 competent cells and cultured in LB solid plate medium for 24 hours in an inversion manner in a 37 ℃ incubator, single colony expansion culture was selected, protein concentration was measured after IPTG induction expression, bacterial collection ultrasonic disruption and centrifugation, ni-NTA chromatographic column purification, ultrafiltration desalination, ultrafiltration concentration and filtration sterilization, and 66NC protein and 58F protein were obtained respectively and stored at-80 ℃ for standby.

2.6 Preparation of candidate subunit vaccine

The immune group is respectively emulsified with 89AP, 89PA, 66NC, 66NC+58F and Ag85B+P22 respectively and MONTANIDE ISA 61 VG adjuvant at a ratio of 1:1.2, and the control group is respectively emulsified with equal volume of sterile PBS and MONTANIDE ISA 61 VG adjuvant at a ratio of 1:1.2. Oscillating and emulsifying at room temperature until low-speed centrifugation is carried out, and not layering, sucking emulsified liquid to the water surface, wherein the liquid drops are round and not immediately dispersed, thus the emulsification is successful.

2.7 Immunization of mouse groups and candidate subunit vaccines

21C 57BL/6 female mice at 6 weeks of age were randomly divided into 7 groups, each group being 3 replicates, and the immunized group was 89AP immunized group, 89PA immunized group, 66NC immunized group, 66NC+58F immunized group, ag85B+P22 immunized group, PBS control group. The blank group did not perform any experimental operations. The immunized group was inoculated with 50. Mu.g (100. Mu.L) of the emulsified corresponding protein per mouse, and the PBS control group was injected with 100. Mu.L of volume-emulsified PBS. Each mouse was immunized twice by back multipoint subcutaneous injection, 3W of each immunization interval. The mouse spleen lymphocytes were isolated after 3W mice were subjected to IFN-. Gamma.ELISPOT experiments.

2.8 Preparation of mouse spleen lymphocytes

(1) Instruments such as copper mesh, ophthalmic scissors, ophthalmic forceps, tissue forceps, surgical knife and the like which are cut into square shapes with 300 meshes are prepared in advance, and autoclaved in advance for later use.

(2) Mice were sacrificed by neck removal from the two-way 3W and immersed in 75% alcohol for 60: 60 s.

(3) Dissecting the mice in an ultra-clean workbench, separating spleens of the mice, placing the spleens on a copper mesh prepared in advance, placing the copper mesh on a culture dish in a suspension manner, grinding by using a5 mL sterile syringe piston, and simultaneously adding a total of 5 mL homogenate washing liquid to wash the copper mesh at the grinding position in a divided manner, so that cells after the spleens are sufficiently ground are all collected in the culture dish through the copper mesh.

(4) The liquid in the petri dish was collected into a 15 mL centrifuge tube and centrifuged at 450 Xg for 5min at room temperature.

(5) The supernatant is discarded, the sediment is added with 5 mL of the erythrocyte lysate which is preheated in a 37 ℃ water bath in advance, and the mixture is left to stand for 5min after being fully resuspended and centrifuged for 5min at room temperature of 450 Xg.

(6) The supernatant was discarded, and 5mL of sterile PBS containing 1% of green streptomycin was added to the pellet and placed in a 37 ℃ water bath in advance, and after sufficient re-suspension, the pellet was centrifuged at room temperature 450 Xg for 5min.

(7) The supernatant was discarded and the pellet was resuspended in 4 mL sample dilution.

(8) 4 ML cell separation liquid is added into a 15 mL centrifuge tube, the sample dilution liquid after the previous step of re-suspension is slowly added onto the cell separation liquid along the tube wall to form two layers of separation liquid surfaces, and the centrifugation is carried out at room temperature of 450 Xg for 10 min.

(9) After centrifugation, it was seen that a milky white layer of cyclic lymphocytes was formed in the centrifuge tube, and lymphocytes were carefully aspirated into a 15 mL centrifuge tube using a 2mL syringe, 5mL cell wash was added, and centrifuged at 450 Xg at room temperature for 5 min.

(10) The supernatant pellet was discarded, and the washed cells were resuspended in 5 mL cell wash solution and centrifuged at 450 Xg at 5 min.

(11) The supernatant pellet was discarded and the washed cells were resuspended in 5 mL sterile PBS containing 1% of streptomycin and centrifuged at 5 min at 450 Xg.

(12) The supernatant is discarded, the sediment is remained, and the cell sediment is resuspended by using the RPMI 1640 complete culture medium which is preheated in a water bath kettle at 37 ℃ in advance, thus obtaining the separated spleen lymphocytes.

2.9 ELISA spot (ELISPOT) assay

Aseptic manipulation:

(1) Add 200. Mu.L of RPMI 1640 medium to activate IFN-. Gamma.ELISPOT pre-coated plate per well, leave it stand for 10 min and dry the liquid in the wells.

(2) The spleen lymphocytes from mice were adjusted to a concentration of 5X 105 cells/100. Mu.L using RPMI 1640 medium, and 100. Mu.L of the cell suspension was added to the activated pre-coated plate. The positive control wells, the negative control wells and the experimental wells are the corresponding groups of mouse spleen lymphocytes, and the blank control wells are 100 mu L of RPMI 1640 complete medium without cells.

(3) 10. Mu.L of stimulus (89 PA, 66NC, 66NC+58F or Ag85B+P22) was added to the wells, respectively. Positive control wells were positive stimulator PMA+ Ionomyci, negative control wells were PBS, experimental wells were protein at a final concentration of 10 μg/mL, and blank control wells were RPMI 1640 medium.

(4) The plate cover was covered and the stimulus was incubated 48 h in a 37 ℃ 5% CO2 incubator.

No aseptic manipulation is required:

(5) The liquid in the wells was dried and 200. Mu.L of pre-chilled ddH2O was added to each well and the cells were lysed at 4℃at 10 min.

(6) The liquid in the wells was dried, 260. Mu.L of 1 XWashing Buffer Washing solution was added to each well, and the mixture was allowed to stand for 1 min to obtain a liquid. Repeated 6 times.

(7) Mu.L of 1X Biotinylated Antibody working solution was added to each well and incubated at 37℃in an incubator for 60 min.

(8) Repeating the step (6).

(9) Mu.L of 1 Xstrepitavidin-HRP working solution was added to each well and incubated at 37℃in an incubator at 60 min.

(10) Repeating the step (6).

(11) Taking down the base, gently flushing the bottom surface of the membrane and the base by using ddH2O, gently sucking residual water trace by using water-absorbing paper after water is fastened, and closing the base.

(12) 100. Mu.L of the AEC color development solution prepared now was added per well protected from light, and incubated at 37℃for 30 min ℃protected from light.

(13) The liquid in the hole was dried, and the front and back sides of the membrane and the base were gently rinsed 3 times with ddH2O to terminate the color development.

(14) After air drying, the reading was performed with an enzyme-linked immunosorbent assay.

2.10 Statistical analysis

The experimental data of each group were analyzed by single factor variance using GRAPHPAD PRISM 8.0.0 software, ns: P >0.05, P <0.01, P <0.001.

3. Experimental results

3.1 Construction and identification of four recombinant plasmids

The p22 and map1609c genes were PCR amplified. As a result, as shown in FIG. 2, 2 specific bands of about 636 bp and 870 bp, respectively, appeared, which were consistent with the expected size of the target gene. And respectively connecting the PCR products with the digested pET-28a-3527N-1609C-3527C and pET-28a-3527N-P22-3527C vectors to obtain 2 recombinant plasmids. The recombinant plasmid which is successfully constructed is identified by PCR and sequencing, and the result is shown in figure 3, and the specific band is consistent with the size of the target gene. Indicating that the recombinant plasmids pET-28a-3527N-1609C-P22-3527C and pET-28 a-3527N-P22-1609C-3527C were successfully constructed.

3.2 Expression and identification of 89AP and 89PA proteins

The identified correct recombinant plasmids pET-28a-3527N-1609C-P22-3527C and pET-28a-3527N-P22-1609C-3527C are respectively transformed into E.coil BL21 competent cells to obtain recombinant bacteria 89AP_BL21 and 89PA_BL21, and protein expression conditions are identified through SDS-PAGE and Western blot after IPTG induction expression, and the result is shown in figure 4, and the molecular weight of 89AP and 89PA is 89.2 kDa.

3.3 Affinity chromatography purification of 89AP and 89PA proteins

The results of the purification of the protein detected by SDS-PAGE after the 2 recombinant bacteria are respectively subjected to activation, expansion culture and IPTG induction, ultrasonic treatment, denaturation treatment and renaturation treatment and purification by Ni column affinity chromatography are shown in figure 5. And (3) ultrafiltering, desalting and concentrating the obtained eluent containing the purified protein to obtain high-purity proteins 89AP and 89PA, wherein the molecular weight of the high-purity proteins is 89.2 kDa, and the amino acid sequences of the high-purity proteins are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

3.4 Affinity chromatography purification of 66NC and 58F proteins

Recombinant bacteria constructed by Shao Mingzhu are used for expressing 66NC protein, the protein purification condition is detected by SDS-PAGE after Ni column affinity chromatography purification, the result is shown in figure 6, the high-purity 66NC protein is obtained, the size is 66 kDa, and the amino acid sequences are respectively shown in SEQ ID NO.3.

The recombinant bacteria constructed by Chen Fanre are used for expressing 58F protein, the purification condition of the protein is detected by SDS-PAGE after purification by Ni column affinity chromatography, the result is shown in figure 7, 58F protein with high purity and the size of 58 kDa is obtained, and the amino acid sequences of the 58F protein are shown in SEQ ID NO.4 respectively.

3.5 EISPOT Experimental screening of candidate antigens

To screen candidate antigens capable of inducing a strong immune response, mice were immunized by subcutaneous multipoint injection after emulsification with the monoide ISA 61 VG adjuvant with 89PA, 66NC, 66nc+58f and ag85b+p22 (wherein the amino acid sequences of Ag85B and P22 are shown as SEQ ID No.5, 6 respectively) and isolated from spleen lymphocytes of the mice after 3. 3W reexposure to carry out IFN- γ ELISPOT experiments. The experimental results are shown in FIG. 8, in which the spleen lymphocytes of 89 AP-immunized mice secrete significantly higher amounts of IFN-gamma cells than the other groups (P < 0.01), indicating that 89AP recombinant protein is able to induce a strong IFN-gamma immune response.

EXAMPLE 2 evaluation of immunogenicity of candidate subunit vaccines for 89AP

1. Experimental materials

1.1 Experimental animal

48C 57BL/6 female mice of 6 weeks old were purchased from Experimental animal technology Co., ltd, fed to the Harbin veterinary institute of research center, and later study was conducted after approval by the animal ethical committee of Harbin veterinary institute of agricultural sciences (HVRI-IACUC-230220-01-GR).

1.2 Main reagent

1.3 Principal solution

CBS solution 0.05M sodium carbonate solution. I.e. 1.59 g sodium carbonate and 2.93 g sodium bicarbonate were dissolved in 1L de-ionized and stored at a pH of 9.6,4 ℃.

PBST solution, PBS containing 0.05% Tween-80, was stored at room temperature.

FACS PBS containing 0.05% sodium azide and 1% BSA was stored at 4℃in the dark.

2. Experimental method

2.1 Preparation of candidate subunit vaccine

Removing endotoxin, namely removing protein endotoxin by using ETERASER HP high-efficiency endotoxin removal kit.

(1) And (3) activating resin, namely vertically fixing a pre-packed column in the kit, adding 5 mL pre-cooled regeneration buffer solution, flowing through the buffer solution at the speed of 0.25 mL/min, and repeating the steps twice.

(2) Balancing resin, namely adding 6mL precooled balancing buffer solution into the activated resin, and flowing through the buffer solution at the speed of 0.5 mL/min, and repeating the steps twice.

(3) Removing endotoxin, namely adding 1.5 mL protein into the equilibrated column, continuously adding 1.5 mL protein after finishing running at the speed of 0.25 mL/min, and collecting effluent liquid, namely the endotoxin-removing protein sample.

89AP immunization group, in which 50 μg/100 μl of 89AP protein was emulsified with MONTANIDE ISA 61 VG adjuvant 1:1.2, 66NC immunization group, in which 50 μg/100 μl of 66NC protein was emulsified with MONTANIDE ISA 61 VG adjuvant 1:1.2, PBS control group, in which equal amount of sterile PBS was emulsified with MONTANIDE ISA 61 VG adjuvant 1:1.2, and blank group was not treated.

2.2 Grouping and immunization of animals

48 6-Week-old C57BL/6 female mice were randomly divided into 4 groups, namely 89AP immunized group, 66NC immunized group, PBS control group, blank group, and 12 replicates per group. The immunized group was inoculated with 50. Mu.g (100. Mu.L) of emulsified 89AP protein or 66NC protein per mouse, and the PBS control group was injected with 100. Mu.L of emulsified PBS. The blank group did not perform any experimental operations. Each mouse was immunized twice by back multipoint subcutaneous injection, 3W of each immunization interval. The immunization scheme is shown in FIG. 9. Animal experiments have been approved by the animal ethics committee of the Harbin veterinary institute of sciences of China (HVRI-IACUC-230220-01-GR).

2.3 Antibody detection

(1) 6 Mice were collected from the tail veins of each of the first immunization groups 2W, 4W, 6W, 8W, and 10W, and after incubation at 37℃for 1h, the serum was collected by centrifugation at 4℃for 1h and at 3 rpm, and stored at-20 ℃.

(2) Coating 66NC and 89AP proteins were diluted with CBS buffer to a final concentration of 5. Mu.g/mL, respectively, and added as coating antigen to 96-well plates at 100. Mu.L per well at 4℃overnight.

(3) Washing, namely buckling and drying the liquid in the holes, adding 260 mu L of PBST into each hole for washing, and buckling out the liquid in the holes after washing. Repeated 5 times.

(4) Blocking 100. Mu.L of PBST-solubilized 5% skim milk was added to each well, blocking 2 h at 37 ℃.

(5) Repeating the step (3).

(6) Serum samples were diluted 1:200, 1:400, 1:800, 1:1:600, 1:3 200, 1:6 400, 1:12 800, 1:25 600, 1:51 200, 1:102 400, 1:204 800, 1:409 600-fold with PBST, incubated at 100 μl per well, 37 ℃ for 1 h.

(7) Repeating the step (3).

(8) Secondary antibody dilution with PBST at Goat anti mouse IgG/IgM labeled with HRP, dilution 1:400.00, incubation 1:1 h at 37 ℃.

(9) Repeating the step (3).

(10) Color development 100. Mu.L of TMB color development solution was added to each well and developed 15 min at 37℃in the dark.

(11) Read value 100. Mu.L of 2M sulfuric acid stop solution was added to each well, and the absorbance was measured at OD450 nm using a microplate reader. The titers of IgG and IgM are the maximum serum dilutions at positive OD450 nm/OD450 nm values of > 2.0.

2.4 ELISPOT experiments

Preparation of spleen lymphocytes from mice with the aid of the double-exempt 3W is described in example 1.2.8. IFN-. Gamma.IL-4 ELISPOT detection methods reference example 1.2.9.

2.5 Serum cytokine detection

(1) Blood was collected from the eyeballs of each group of mice after the second 3W days, and serum was collected.

(2) Diluted 1 Xcapture anti-ibody, 100. Mu.L/well, was added to a 96-well plate and coated overnight at 4 ℃.

(3) The liquid in the wells was dried, washed with 260. Mu.L/well PBST and repeated 5 times.

(4) Diluted 1 XELISA/ELISPOT reagent, 100. Mu.L/well, was added and blocked at room temperature of 1 h.

(5) Repeating the step (3).

(6) Standard powder was dissolved with ddH2O and diluted 2-fold to obtain 8 standards of different concentrations. 100. Mu.L of serum was added to each well of the experimental well, 100. Mu.L of a gradient concentration of standard was added to each well of the standard, 2-well replicates were made for each concentration of standard, and 100. Mu.L of 1 XELISA/ELISPOT reagent was added to each well of the blank wells, and incubated at room temperature for 2 h.

(7) Repeating the step (3).

(8) Mu.L of 1X detection antibody was added to each well and incubated at room temperature for 1:1 h.

(9) Repeating the step (3).

(10) Mu.L of 1 Xenzyme was added to each well and incubated at room temperature for 30 min.

(11) Repeating the step (3).

(12) 100 Mu L of TMB color development liquid is added to each hole in a dark place, and color development is carried out at 37 ℃ in a dark place for 15 min.

(13) The color development was stopped by adding 100. Mu.L of 2M sulfuric acid stop solution to each well, and the light absorption was read at OD450 nm with a microplate reader.

2.6 Intracellular cytokine detection

Aseptic manipulation:

(1) Stimulation and culture of splenic lymphocytes 2 mL cells of 1X 106 cells/mL of mouse splenic lymphocytes were added per well in a 6-well cell culture plate. Different stimulators were added according to the experimental group as 89AP immune group and 66NC immune group respectively with 89AP and 66NC proteins with final concentration of 20 μg/mL, PBS control group with sterile PBS with the same volume as the immune group, and blank control group with RPMI 1640 medium with the same volume as the immune group. After gentle shaking mixing, the plate was covered and stimulated 12 h in a 37 ℃ 5% CO2 incubator.

(2) 1.3 Mu L BD GolgiStop and 2 mu L BD GolgiPlug were added to each well, and after thoroughly mixing, the wells were covered with a plate cover and blocked in a 37℃5% CO2 incubator by 3.5 h.

No aseptic manipulation is required:

(3) After gently suspending the cell pellet, transfer to a 15 mL centrifuge tube and centrifuge 5 min at 450 Xg.

(4) Cell pellet was retained, cells were resuspended by addition of 1mL FACS, centrifuged at 450 Xg for 5min, and repeated 2 times.

(5) Cell pellet was retained and resuspended with 100 μl FACS and transferred to labeled tubes of CD4 and CD8, respectively, 50 μl per tube. 1. Mu.L of Anti-Mouse CD4 antibody was added to the CD 4-labeled tube, 1. Mu.L of Anti-Mouse CD8 antibody was added to the CD 8-labeled tube, and after mixing, the mixture was incubated on ice in the absence of light for 30 min.

(6) 1 ML FACS was added per tube, centrifuged at 450 Xg for 5 min, the supernatant was discarded, and the pellet was repeated 2 times.

(7) Each tube was added with 250. Mu. L Fixation solution, the pelleted cells were resuspended well, incubated on ice for 30min in the dark, centrifuged at 450 Xg for 5: 5 min, and the cell pellet was retained.

(8) Cells were washed by adding 1 mL of 1 XBD Perm Buffer per tube, resuspended well, centrifuged at 450 Xg for 5 min, and cell pellet was retained and repeated 2 times.

(9) 50. Mu.L of 10 XBD Perm Buffer was added to each tube, followed by 1. Mu.L of Anti-Interferon gamma, anti-Mouse IL-17A, and incubation in the dark on ice for 30min was performed after full resuspension.

(10) Repeating the step (8).

(11) Cells were resuspended in 300 μl PBS per tube, detected using a multiparameter flow cytometer and analyzed for results.

2.7 Statistical analysis

3. Experimental results

3.1 Antibody detection results

To evaluate the effect of candidate subunit vaccines 89AP and 66NC on antibody levels, the change in antibody titers of IgG, igM was detected starting at prime 2W. The results as shown in figure 10 indicate that both 89AP and 66NC can induce higher levels of antibody.

3.2 ELISPOT test results

To compare the immune levels of candidate protein 89AP and 66NC subunit vaccine, spleen lymphocytes from the 3W-di-immune mice were isolated and IFN-. Gamma.ELISPOT and IL-4 ELISPOT experiments were performed. The results showed that the spleen lymphocytes from 89AP immunized mice secreted significantly higher amounts of IFN-. Gamma.than 66NC immunized mice, but no significant difference (P > 0.05), as shown in FIG. 11, and that 89AP immunized mice significantly different (P < 0.01) from PBS control and blank, as shown in FIG. 12, the spleen lymphocytes from 89AP immunized mice secreted significantly higher amounts of IL-4 than 66NC immunized mice, PBS control and blank (P < 0.01). The results show that the candidate subunit vaccine 89AP can induce IFN-gamma and IL-4 immune responses at higher level, and is a candidate antigen with better immune effect.

3.3 Serum cytokine detection results

To compare the difference in serum cytokine levels induced by candidate subunit vaccines 89AP and 66NC, serum was collected by sampling each mouse in each group at the time of 3W for the second immunization, and the IL-17A and IFN- γ levels in the serum of the mice were detected by ELISA. The results are shown in FIG. 13, where the serum cytokines IL-17A and IFN-gamma secreted by the 89AP immune group were significantly higher than those of the 66NC immune group and PBS control group (P < 0.01), indicating that the candidate subunit vaccine 89AP was better able to stimulate the release of IL-17A and IFN-gamma in the serum of mice.

3.4 Intracellular cytokine detection results

To evaluate the induction of immune responses by candidate subunit vaccine 89AP, the levels of IFN- γ, IL-17A and IL-2 secreted by cd4+ T and cd8+ T cells were examined using ICS, and the results, as shown in fig. 14, showed that the 89AP immunized group, cd4+ T and cd8+ T cells, secreted IFN- γ, IL-17A and IL-2 significantly higher than the PBS control group and blank group (P < 0.01), slightly higher than the 66NC immunized group, but not significantly different (p=0.1), indicated that candidate subunit vaccine 89AP could better induce high levels of IFN- γ, IL-17A and IL-2 secreted by cd4+ T and cd8+ T cells.

EXAMPLE 3 evaluation of the immune Effect of 89AP candidate subunit vaccine

1. Experimental materials

1.1 Experimental animal

24C 57BL/6 female mice of 6 weeks old were purchased from Experimental animal technology Co., ltd, fed to the Harbin veterinary institute of research center, and later study was conducted after approval by the animal ethical committee of Harbin veterinary institute of agricultural sciences of China (HVRI-IACUC-230220-01-GR).

1.2 Main reagent

2. Experimental method

2.1 Grouping and infection of animals

24 6 Week old C57BL/6 female mice were randomly divided into 4 groups of 6 animals each, 89AP immunized group, 66NC immunized group, PBS control group and blank control group. Vaccine immunized mice were prepared according to the method of example 2.2. MAP K-10 strain at a concentration of 1X 109 CFU/mL was intraperitoneally injected into infected mice after 3W post-priming wash with sterile PBS, and 100. Mu.L of each mouse was injected except for the placebo group.

2.2 Serum cytokine detection

Serum was collected by ocular blood collection at infection 2W and serum cytokine detection method was described in example 2.5.

2.3 Intracellular cytokine detection

Serum was collected by ocular blood collection at the time of infection 2W, and intracellular cytokine detection method was described in example 2.6.

2.4 Pathology and histopathology observations

The liver and small intestine of mice after MAP K-10 infection of 2W were isolated in an ultra clean bench, photographed to record macroscopic lesions of the viscera and scored for pathology. Taking tail leaves and ileum segments of mouse livers, placing the tail leaves and ileum segments into 30 mL tissue fixing solution for fixing more than 48 h, and sending the tail leaves and ileum segments to a pathology laboratory of Harbin veterinary institute of China academy of agricultural sciences for preparing pathological sections, and performing HE and acid-fast staining. Histopathological scoring was performed by observing histopathological changes. The pathological scoring rules are as follows, the percentage of surface nodules of liver is 0 = none, 1= <1%, 2= > 1% and <3%, 3= > 3% and <5%, and the percentage of surface nodules of small intestine is 0 = none, 1 = 1. Histopathological scoring of liver and small intestine was performed according to granulomatous area percentage 0 = none, 1= <10%, 2= ∈10% and <15%, 3= ∈15% and <20%, 4= ∈20% and <30%.

2.5 Colony field planting

After MAP K-10 infection of mice 2W, the mice were sacrificed by cervical removal, the livers and small intestines of the mice were isolated in an ultra clean bench and placed in three dishes each containing 2mL mycobacteria complete medium, and were piston milled using a copper mesh and 5mL syringe. 200. Mu.L of the ground stock solution was added dropwise to a 7H10 solid medium (containing 50. Mu.g/mL of sodium nalidixic acid and 50. Mu.g/mL of vancomycin hydrochloride) and cultured in a 37℃incubator for 4 or more W hours, and colony growth conditions on the medium were observed to perform colony counting.

2.6 Statistical analysis

3. Experimental results

3.1 Serum cytokine detection results

To compare the difference in serum cytokines between the 89AP immune group and the 66NC immune group after infection 2W, the analysis was performed using ELISA assay. The results are shown in FIG. 15, where IL-17A and IFN-gamma secretion levels were significantly higher in the 89AP immunized mice than in the 66NC immunized and PBS control (P < 0.01). The results show that the candidate recombinant subunit vaccine 89AP can stimulate the release of IL-17A and IFN-gamma in the serum of mice, and induce stronger Th1 type and Th17 type immune responses.

3.2 Intracellular cytokine detection results

In order to differentiate serum cytokines between the 89AP immunized group and the 66NC immunized group after infection with 2W, the levels of IFN-gamma, IL-17A and IL-2 secreted by CD4+ T and CD8+ T cells were examined using ICS, and as shown in FIG. 16, the IFN-gamma, IL-17A and IL-2 secreted by CD4+ T and CD8+ T cells of the 89AP immunized group were significantly higher than those of the 66NC immunized group, PBS control group and blank group (P < 0.01). The results show that the candidate recombinant subunit vaccine 89AP can stimulate the release of IL-17A and IFN-gamma in the serum of mice, and induce stronger Th1 type and Th17 type immune responses.

3.3 Pathology and histopathology observations

To evaluate the immunoprotection effect of candidate recombinant subunit vaccine 89AP on mice, mice were dissected after MAP infection of mice 2W, and pathology and histopathology analysis was performed on the liver and small intestine of the mice. The pathology results are shown in fig. 17, and the liver lesions of 89AP immunized group were significantly decreased and the differences were very significant (P < 0.01) when MAP-infected mice 2W were compared to PBS control group and 66NC immunized group. The 89AP immunized group also had reduced intestinal lesions, but the difference was not significant compared to the 66NC immunized group (P > 0.05), and the difference was significant compared to the PBS control group (P < 0.05). Histopathological results as shown in fig. 18, extensive hepatocyte necrosis and granuloma occurred in all immunized groups.

3.4 Colony colonization results

To evaluate the protective effect exerted by 89AP recombinant subunit vaccine during infection, liver and small intestine of mice infected with 2W of 89AP immunized group, 66NC immunized group and PBS control group were taken for colony count and acid-fast staining of liver. The results are shown in figure 19, in which the number of liver lotus bacteria in the 89AP immunized group after 2W infection is significantly lower than that in the other groups, the difference is significant (P < 0.05), and in which the number of small intestine lotus bacteria in the 89AP immunized group after 2W infection is significantly lower than that in the other groups, the difference is significant (P < 0.05). The 89AP recombination candidate subunit vaccine can obviously reduce the lotus number in the liver and intestinal tract of mice after MAP infection, and has a certain protection effect on resisting MAP infection.

EXAMPLE 4 guinea pig model evaluation of the immune Effect of 89AP candidate subunit vaccine

1. Experimental materials

1.1 Experimental animal

Less than 300g of clean grade guinea pigs without specific pathogens were purchased from beijing velariwa laboratory animal technologies limited. Are raised in the laboratory animal center of the Harbin veterinary institute. The animal experiments have been followed by approval by the animal ethics committee of the Harbin veterinary institute of agricultural sciences, china (HVRI-IACUC-231211-03-GR).

1.2 Main reagent

The allergen is purified bovine tuberculin 1 mg/mL and purified paratuberculin 1 mg/mL, which are obtained from Shanghai Chengrui Biotechnology Co.

2. Experimental method

2.1 Preparation of candidate subunit vaccine

The MAP K-10 strain stored in the laboratory was cultured to the logarithmic growth phase by the method of reference example 1.2.1, the bacterial pellet was collected by centrifugation at 10min at 3 rpm, the bacterial pellet was resuspended in sterile ddH2O according to the bacterial count, and then centrifuged, and the medium was washed off and repeated three times. Re-suspending the bacterial precipitate with 10mL sterile ddH2O, inactivating the bacterial precipitate in a constant temperature water bath at 80 ℃ for 30min, inoculating the bacterial liquid after partial inactivation into a mycobacteria ready-to-use complete medium (containing 1mg/mL mycobacteriin), and culturing to check whether the inactivation is complete. The inactivated bacterial liquid is centrifuged at 3.500 rpm for 10min, the supernatant is discarded, the precipitated bacterial cells are spread in an 80 mm sterile plastic culture medium, and the bacterial cells are kept stand in an ultra clean workbench until the bacterial cells are completely dried. Scraping the dried fungus, weighing K-10 inactivated fungus by using an electronic analytical balance, recording, preparing a fungus suspension of 5 mg/mL by using PBS, and emulsifying with MONTANIDE ISA 61 VG adjuvant in a ratio of 1:1 to ensure that the concentration reaches 2.5 mg/mL.

Candidate subunit vaccines were prepared by the method of reference example 2.1 and stored at 4 ℃ for use after preparation.

BCG vaccine Tokyo 172 strain standard (BCG) was stored by the laboratory.

2.2 Grouping and immunization of animals

Guinea pigs were randomly divided into 5 groups, 89AP immunized group, 66NC immunized group, PBS control group, 3-5 replicates per group, and 2 replicates per group of paratuberculosis positive control group and tuberculosis positive control group. The immunized group was inoculated with 50. Mu.g (100. Mu.L) of emulsified protein 89AP or 66NC per mouse, the PBS control group was injected with 100. Mu.L of emulsified PBS per mouse, the paratuberculosis positive control group was inoculated with MAP K-10 inactivated vaccine per subcutaneous, and the tuberculosis positive control group was immunized with BCG per subcutaneous (OD 600. Apprxeq.1.0, 100. Mu.L of each immunization).

2.3 Antibody detection

Guinea pig serum was collected by cardiac blood sampling at 2W, 4W, 6W, 8W, respectively, after the first immunization, and the antibody detection method was described in example 2.3.

2.4 Inoculation of allergens

Diluting purified bovine tuberculin and purified tuberculin with PBS to 40 μg/mL for use, dehairing the two sides of the back of guinea pigs subjected to the two-stage treatment of 2W, sterilizing the hairless area with 75% alcohol, and injecting allergen at different points. The left side is divided into an injection point a and an injection point b from the head to the tail, 50 mu L of purified bovine tuberculin and 50 mu L of PBS are respectively injected intradermally, the right side is divided into an injection point c and an injection point d from the head to the tail, and 50 mu L of purified auxiliary tuberculin, 50 mu L of 89AP protein or 50 mu L of 66NC protein are respectively injected intradermally.

2.5 Allergic reaction result determination

After intradermal injection, interval 48 h was recorded using vernier calipers to measure the diameter of the skin's appearance response (red, swollen) at each injection site, respectively.

2.6 Statistical analysis

Statistical analysis was performed on each set of experimental data using GRAPHPAD PRISM 8.0.0 software.

3. Experimental results

3.1 Antibody detection results

To evaluate the humoral immune response induced by candidate subunit vaccine 89AP in guinea pigs, the change in antibody titer of IgG was detected beginning at first 2W. As the results in fig. 20 show, 89AP was able to induce guinea pigs to produce high levels of antibodies, with no significant difference from the candidate subunit vaccine 66 NC.

3.2 Allergic reaction detection results

In order to study whether the quarantine of intradermal allergies of tuberculosis and paratuberculosis is affected after the recombinant antigen 89AP is immunized, 89AP is immunized with guinea pigs, and the immunized guinea pigs of candidate subunit vaccine 66NC, MAP K-10 inactivated vaccine and BCG vaccine are used as controls, purified bovine tuberculin and avian tuberculin are inoculated in the skin respectively, and the allergies are detected after 48 h is inoculated. The results showed that MAP K-10 inactivated vaccine immunized guinea pigs received bovine tuberculin (10.71.+ -. 2.35 mm) and paratuberculin (6.69.+ -. 1.13 mm), BCG vaccine immunized guinea pigs received bovine tuberculin (12.96.+ -. 0.01 mm) and paratuberculin (9.5.+ -. 0.57 mm) all showed strong allergic reactions, 89AP, 66NC immunized groups and PBS control groups showed no allergic reactions to bovine tuberculin and paratuberculin, but 89AP immunized groups showed strong allergic reactions to 89AP protein stimulation (5.87.+ -. 0.84 mm) and 66NC immunized groups to 66NC protein stimulation (5.57.+ -. 0.37 mm) (FIG. 21). The results show that the 89AP and 66NC candidate subunit vaccines have immune effects on guinea pigs and do not interfere with the skin test allergy quarantine of bovine tuberculosis and paratuberculosis.

Claims

1. The paratuberculosis subunit vaccine is characterized by comprising 89AP recombinant antigen, wherein the 89AP recombinant antigen is obtained by fusion expression of MAP3527, MAP1609c and p22 genes of paratuberculosis mycobacterium (Mycobacterium avium subsp. Paratuberculosis, MAP), and the amino acid sequence of the 89AP recombinant antigen is shown as SEQ ID NO.1.

2. The paratuberculosis subunit vaccine of claim 1, wherein the 89AP recombinant antigen is obtained by a prokaryotic expression system.

3. The paratuberculosis subunit vaccine of claim 2, wherein the 89AP recombinant antigen is obtained by transferring a recombinant plasmid pET-28a-3527N-1609C-P22-3527C into escherichia coli BL21 for culturing and expressing, purifying by Ni column affinity chromatography, and constructing the recombinant plasmid pET-28a-3527N-1609C-P22-3527C by the following method:

(1) PCR amplification of p22 Gene

Amplifying p22 gene with p22-F/R by using extracted MAP K-10 strain whole genome DNA as template, recovering the PCR product after identification correctly by using a gel recovery kit, measuring the concentration, and freezing and preserving in a refrigerator at-20 ℃;

p22-F:GGCGGCGGAcaagcttgcTGCTCGTCGGGCTCCAAGCA p22-R:GGCGGCGGTgcaagcttgCGAGCTCACCGGGGGCTTGG

(2) Single enzyme digestion of pET-28a-3527N-1609C-3527C vector

(3) Ligation of the cleavage vector with the PCR product

Connecting the pET-28a-3527N-1609C-3527C digestion products with the P22 product to construct a recombinant plasmid pET-28a-3527N-1609C-P22-3527C;

(4) Conversion of ligation products

Adding the connected products into E.coil DH5 alpha competent cells respectively, uniformly mixing, carrying out ice bath for 30min, immediately carrying out heat shock for 30-45s in a42 ℃ water bath, immediately carrying out ice bath for 2min, adding 500 mu L of LB liquid medium, recovering 60min at 37 ℃ and 180rpm, centrifuging for 5min at 3 500rpm, discarding the supernatant, resuspending the precipitated thalli with 100 mu L of LB liquid medium, coating on LB solid flat plate medium containing 50 mu g/mL of kanamycin, culturing for 24h at 37 ℃ in a temperature box, obtaining single colony, picking single colony with good growth state, inoculating the single colony into 5mL of LB liquid medium containing 50 mu g/mL of kanamycin, culturing at 37 ℃ and 180rpm, centrifuging for 5min at 3 500rpm, extracting plasmids by using a plasmid small extraction kit, carrying out PCR and sequencing to identify recombinant plasmids pET-28a-3527N-1609C-P22-3527C;

(5) Induction of 89AP protein expression

5.1 Construction of the Strain of overexpressed protein BL21

Transferring 5-10 mu L of the recombinant plasmid pET-28a-3527N-1609C-P22-3527C identified correctly into 50 mu L of E.coilBL21 competent cells;

5.2 Induction of expression of recombinant proteins

Selecting BL21 single colony in LB solid medium, inoculating in 5mL LB liquid medium containing 50 mug/mL kanamycin, culturing at 37 ℃ at 180rpm for 24h, transferring 100 mug bacterial liquid into 10mL LB liquid medium containing 50 mug/mL kanamycin, continuously culturing until OD ₆₀₀ = 0.8, collecting 1mL whole bacterial liquid before induction, preparing sample, preserving, adding IPTG with final concentration of 1mM into the rest bacterial liquid, inducing protein expression at 37 ℃ at 180rpm for 4h, collecting 1mL whole bacterial liquid after induction, preparing sample, preserving;

5.3 identification of recombinant proteins

(6) 89AP protein purification.

4. A paratuberculosis subunit vaccine according to claim 3, wherein said step (6) comprises the steps of:

(1) Selecting identified BL21 single colony from LB solid plate medium, inoculating to 10mL LB liquid medium containing 50 μg/mL kanamycin, culturing at 37 ℃ at 180rpm for 24h, transferring 10mL bacterial liquid to 1L LB liquid medium containing 50 μg/mL kanamycin, continuously culturing until OD600 = 0.8, adding IPTG with final concentration of 1mM at 37 ℃ at 180rpm, inducing protein expression for 4h, centrifuging at 4 ℃ at 8 rpm for 10min, collecting bacterial precipitate, suspending each 1L bacterial precipitate by using 40mL Buffer A, performing ultrasonic crushing for 3s with amplitude of 38%, stopping for 3s, performing effective ultrasonic time for 30min, performing primary centrifugation at 4 ℃ at 3 rpm for 10min after ultrasonic crushing, centrifuging, discarding precipitated fragments, and centrifuging at 10000 rpm for 30min again;

wherein Buffer A is 20mM Tris-HCl containing 150mM NaCl and 10% v/v glycerol, and the pH value is 8.0;

(2) Protein is expressed as inclusion bodies, the sediment after centrifugation is collected, the sediment is resuspended by 80mL Buffer B, the solution is clear by stirring overnight at room temperature by using a rotor, the solution is centrifuged for 30min at 4 ℃ at 10 rpm, the supernatant is collected, the concentration is diluted to be less than 1mg/mL by using Buffer B, the solution is transferred into a dialysis bag, 1L of renaturation solution is used for soaking the supernatant in every 100mL of dialysis bag, after stirring for 18h at 4 ℃, the dialysis bag is replaced into Buffer A, stirring for 18h at 4 ℃, after renaturation is finished, the supernatant in the dialysis bag is taken out, and the supernatant is centrifuged for 30min at 4 ℃ at 12 rpm and collected;

wherein, buffer B is 20mM Tris-HCl containing 150mM NaCl,0.8% w/v SKL and 10% v/v glycerol, and the pH is 8.0;

the renaturation solution is 150mM Tris-HCl containing 0.54g of reduced glutathione, 0.06g of oxidized glutathione, 150mM NaCl and 3% v/v glycerol, and the pH value is 7.5;

(3) Taking 20 mu L of supernatant sample before hanging a column, and preparing a pre-threading liquid;

(4) After 20mL Buffer A flows through the Ni-NAT chromatographic column to balance column materials, the supernatant protein passes through the chromatographic column, the flowing-through liquid is collected, and the column is repeatedly hung for 6 times;

(5) Taking 20 mu L of supernatant sample after column hanging, and preparing a penetrating fluid;

(6) Using 20mL Buffer A to flow through Ni-NAT chromatographic column balance column material, adding 5mL Buffer A heavy suspension column material, taking 20 mu L column material suspension sample, and making resin pass through;

(7) Passing through the columns with 40mL Buffer C,40mL Buffer D,40mL Buffer E,40mL Buffer F,20mL Buffer G,20mL Buffer H streams respectively to wash off the impurity proteins;

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

(8) Passing 20mL of Buffer A through the Ni-NAT chromatographic column to balance the column material, adding 5mL of Buffer A to re-suspend the column material, taking 20 mu L of column material suspension to leave a sample, and preparing the resin after impurity washing;

(9) After the column material is naturally precipitated, avoiding suspending the column material, slowly adding 5mL of Buffer I, standing on ice for 20min, collecting eluent 1 left naturally, adding 5mL of Buffer J, standing on ice for 20min, collecting eluent 2, finally adding 5mL of Buffer K, standing on ice for 20min, collecting eluent 3, and taking 20 mu L of each of the 3 eluents as samples;

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

(10) Passing 20mL of Buffer A through the Ni-NAT chromatographic column to balance the column material, adding 5mL of Buffer A to re-suspend the column material, taking 20 mu L of column material suspension to leave a sample, and preparing eluted resin;

(11) Identifying the collected protein samples by SDS-PAGE gel electrophoresis;

(12) According to the analysis result of SDS-PAGE gel electrophoresis, collecting protein eluent, ultrafiltering and desalting by using a 30kDa ultrafiltration and concentration centrifuge tube at 4 ℃ and 3 rpm, concentrating the protein eluent to a volume below 1mL, filtering and sterilizing the protein eluent by using a 0.45 mu m filter, and measuring the protein concentration, and preserving the protein eluent at-80 ℃ for later use.

5. The paratuberculosis subunit vaccine according to claim 1, the vaccine is characterized by further comprising an adjuvant.

6. The paratuberculosis subunit vaccine of claim 5, wherein the adjuvant is a MONTANIDE ISA61VG adjuvant.

7. The paratuberculosis subunit vaccine of claim 6, wherein the vaccine is obtained by emulsifying 89AP recombinant antigen with MONTANIDE ISA61VG adjuvant in a weight ratio of 1:1.2.

8. Use of a paratuberculosis subunit vaccine according to any of claims 1-7 in the manufacture of a medicament for the prevention of paratuberculosis.