A nasal spray subunit vaccine for contagious pleuropneumonia of sheep and a method of preparation

Abstract

This invention belongs to the field of biotechnology, specifically relating to a nasal spray subunit vaccine for ovine infectious pleuropneumonia and its preparation method. It includes a first antigen protein and a second antigen protein, the amino acid sequence of which is shown in SEQ NO:1, and the amino acid sequence of which is shown in SEQ NO:2. The mass ratio of the first antigen protein to the second antigen protein is 1:(1-1.5). This invention [Ca²] + It can stabilize the structure of antigen proteins through ionic bonds, reducing their degradation in nasal mucosal secretions. Tannic acid and Ca²⁺ + When antigen complexes bind, they can form more ordered immunogenic complexes through the synergistic effect of polyphenols, metal ions, and proteins, making some antigens particulate and enhancing the uptake and processing efficiency of antigen-presenting cells. PDA can encapsulate the complex and use its biomimetic adhesion properties to form a firm attachment in the moist environment of the nasal mucosa, prolonging the antigen exposure time and promoting the continuous production of antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nasal spray ovine infectious pleuropneumonia subunit vaccine and its preparation method. Background Technology

[0002] Contagious pleuropneumonia in sheep poses a serious threat to the sheep farming industry, and vaccination is a key prevention and control measure. Traditional tissue-inactivated vaccines typically involve infecting healthy goats with tissue toxins or bacterial solutions, collecting lung tissue and other tissues from infected goats, removing the trachea and bronchi, and then grinding, filtering, and inactivating the virus. This process is complex, requires stringent standards for laboratory animals, is susceptible to contamination by other pathogens, results in inconsistent quality, and limits large-scale production. For example, early domestically developed aluminum-adjuvant tissue-inactivated vaccines, while showing some immunization effect, had significant drawbacks.

[0003] Currently, whole-cell inactivated vaccines are gradually becoming the mainstream; however, under existing technology, they also face many challenges. Mycoplasma caprineis subsp. caprineis, as the main pathogen, is difficult to cultivate, grows slowly, and has low yield, making the production process of whole-cell inactivated vaccines complex, costly, and lacking in large-scale production capacity. It also poses biosafety risks and significant side effects. For example, although the first domestically developed inactivated vaccine for preventing contagious caprine pleuropneumonia (CPP) using the M1601 strain employs a novel nano-adjuvant, improving safety and immunogenicity, overall production problems remain prominent.

[0004] Subunit vaccines are an important research and development direction. They are prepared by extracting effective antigenic components from pathogens, resulting in high purity and good safety. Through whole-genome, comparative genomics, proteomics, and immunoproteomics analyses of *Mycoplasma caprineis* subspecies *Capreolae*, immunogenic proteins are screened. Factors such as cellular localization, protein hydrophilicity, antigenicity, signal peptides, transmembrane regions, T-cell epitopes, and B-cell epitopes are comprehensively considered, and combinations of immunogenic proteins are classified and screened after in vitro and in vivo experiments.

[0005] Both whole-cell inactivated vaccines and subunit vaccines require traditional subcutaneous or intramuscular injection immunization. This method requires 2-3 people to work together to manually inject each sheep one by one, completing steps such as shaving the injection site, disinfecting with alcohol, inserting the needle, and injecting the medication, which consumes a lot of manpower. Physical restraints during the injection process (such as the pressure of the restraint frame) and needle puncture pain can cause severe stress to the sheep. If the disinfection of the injection site is not done properly, it can easily lead to local infection. Mild cases may result in redness, swelling, induration, and suppuration at the injection site, and the sheep may frequently rub and scratch the affected area due to pain, further aggravating the skin damage. In severe cases, the infection may spread to the subcutaneous tissue or muscle layer, causing a systemic inflammatory response, with symptoms such as fever, depression, and a sharp drop in feed intake. In severe cases, antibiotic treatment is required, which not only increases the cost of raising sheep but may also affect the quality of the mutton due to drug residues.

[0006] Therefore, there is an urgent need for a nasal spray subunit vaccine for infectious pleuropneumonia of sheep that is safer, easier to administer, and has fewer side effects. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to overcome the drawbacks of inactivated tissue vaccines, such as complex processing, susceptibility to contamination, and significant side effects; whole-cell inactivated vaccines, which are difficult to culture and costly to produce pathogens; vaccination impacting sheep health and reproduction; traditional injection immunization, which is labor-intensive and time-consuming, prone to missed or incorrect injections, causing significant stress to sheep, and potentially leading to infection due to improper disinfection, thus increasing costs and drug residue risks. The invention provides a nasal spray subunit vaccine for contagious pleuropneumonia in sheep and its preparation method.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] An antigenic protein combination of Mycoplasma caprineis subspecies of caprine pneumonia, comprising a first antigenic protein and a second antigenic protein.

[0010] The amino acid sequence of the first antigen protein is shown in SEQ NO:1, and the amino acid sequence of the second antigen protein is shown in SEQ NO:2.

[0011] Furthermore, the mass ratio of the first antigen protein to the second antigen protein is 1:(1-1.5).

[0012] A nasal spray subunit vaccine for infectious pleuropneumonia in sheep, comprising a first solution, a second solution, and a third solution;

[0013] The first solution contains the above-mentioned antigen protein combination and Ca²⁺;

[0014] The second solution contains tannic acid;

[0015] The third solution contains polydopamine.

[0016] Furthermore, the first solution comprises 50-100 μg / mL of antigen protein combination, 5-10 mmol / L of calcium chloride, 0.01 mmol / mL of aluminum hydroxide, 8.5 g / L of sodium chloride, and 0.01% by mass of polysorbate, using 0.01 mol / L PBS at pH 7.2-7.4 as the solvent.

[0017] Furthermore, the second solution comprises 0.5-1 mg / mL tannic acid, 80 μg / mL CpG ODN, 8.5 g / L sodium chloride, and 0.005% disodium EDTA, using 0.01 mol / L PBS at pH 6.5-7.0 as the solvent.

[0018] Furthermore, the third solution comprises 1-2 mg / mL of polydopamine and 8.5 g / L of sodium chloride, using 0.01 mol / L PBS at pH 7.2-7.4 as a solvent.

[0019] Furthermore, the first solution, the second solution, and the third solution were sprayed into the nasal cavity of the sheep in a volume ratio of 3:1:1.

[0020] A method for preparing a nasal spray subunit vaccine against contagious pleuropneumonia in sheep, comprising the following steps:

[0021] (1) Preparation of the first solution:

[0022] Antigen protein reconstitution: The freeze-dried first and second antigen proteins were reconstituted at 4°C in a 1:1 mass ratio with 0.01 mol / L PBS at pH 7.2, and the concentration was adjusted to 100 μg / mL. Sodium chloride was then added to bring the concentration to 8.5 g / L.

[0023] Ca² + Complex formation: At 4°C, slowly add 1 mol / L CaCl2 solution dropwise to the antigen-protein combination solution until the final concentration is 5 mmol / L, stirring with a magnetic stirrer while adding. After the addition is complete, continue stirring for 30 min to ensure sufficient ionic bond formation. Then, slowly add aluminum hydroxide suspension dropwise, continuing stirring for 1 h to ensure the antigen is coated with Ca²⁺. + Synergistic adsorption with aluminum hydroxide, and the addition of 0.01% polysorbate yields the first solution;

[0024] (2) Preparation of the second solution:

[0025] CpG ODN was dissolved in 0.01 mol / L sterile PBS solution at pH 6.8 to a final concentration of 80 μg / mL. Tannic acid powder was added to a final concentration of 0.8 mg / mL. The solution was vortexed at room temperature until completely dissolved. Sodium chloride was added to a final concentration of 8.5 g / L. Disodium EDTA (0.005% by mass) was then added to obtain the second solution.

[0026] (3) Preparation of the third solution:

[0027] Polydopamine was added to 0.01 mol / L PBS at pH 7.2-7.4 to a final concentration of 1-2 mg / mL, and sodium chloride was added to a final concentration of 8.5 g / L to obtain the third solution.

[0028] Furthermore, the first and second solutions are sterilized by filtration through a 0.22 μm PES filter membrane; the third solution is sterilized by filtration through a 0.22 μm CA filter membrane.

[0029] The beneficial technical effects of this invention are as follows:

[0030] This invention Ca² + As an electrolyte, tannic acid can stabilize the secondary and tertiary structures of antigenic proteins through ionic bonds, reducing their degradation in nasal mucosal secretions. + When antigen complexes bind, the synergistic effect of polyphenols, metal ions, and proteins forms a more ordered immunogenic complex, causing some antigens to become particulate, enhancing the uptake and processing efficiency of antigen-presenting cells. PDA can encapsulate the complex, utilizing its biomimetic adhesion properties to form a firm attachment in the moist environment of the nasal mucosa, prolonging antigen exposure time and promoting the sustained production of local antibodies. Nasal spray administration has low irritation and mild stress response, is simple to operate without professional skills, and administers medication to a single sheep in just 30-40 seconds, making it suitable for sensitive groups such as lambs and pregnant sheep. Attached Figure Description

[0031] Figure 1 Antigenicity analysis of antigen 1 sequence in this embodiment of the invention;

[0032] Figure 2 Antigenicity analysis of antigen 2 sequence in an embodiment of the present invention;

[0033] Figure 3 Antigenicity analysis of antigen 3 sequence in this embodiment of the invention;

[0034] Figure 4 Antigenicity analysis of antigen 4 sequence in this embodiment of the invention;

[0035] Figure 5 These are SDS-PAGE images of four recombinant antigens from embodiments of the present invention;

[0036] Figure 6 This is a graph showing the reactivity detection results of antibodies generated from four recombinant antigens in embodiments of the present invention;

[0037] Figure 7 This is an indirect immunofluorescence result diagram from an embodiment of the present invention;

[0038] Figure 8 This is a graph showing the results of real-time quantitative PCR detection in an embodiment of the present invention;

[0039] Figure 9 The antibody levels at various time points after immunization with different component vaccines in this embodiment of the invention. Detailed Implementation

[0040] The specific implementation method will be further described below with reference to the accompanying drawings.

[0041] The reagents and instruments used in the following examples are all conventional laboratory reagents and instruments. Example 1

[0042] An antigenic protein combination of Mycoplasma caprineis subspecies of caprine pneumonia was obtained. The screening process for the components of the antigenic protein combination is as follows:

[0043] 1. Antigen sequence analysis

[0044] The antigenicity, hydrophilicity / hydrophobicity, secondary structure, and surface accessibility of the 870 genes encoded by the ATCC 27343 strain were analyzed using DNASTAR. Combined with IEDB antigenic epitope prediction results, four antigenic genes were identified. Figures 1-4 The image shows an antigenicity analysis diagram of four antigen sequences. The amino acid sequences of the antigens are as follows:

[0045] The amino acid sequence of antigen 1 is the sequence of SEQ NO:1 (52K) in the sequence listing.

[0046] The amino acid sequence of antigen 2 is the sequence of SEQ NO:3 (95K) in the sequence listing.

[0047] The amino acid sequence of antigen 3 is the sequence of SEQ NO:4 (73K) in the sequence listing.

[0048] The amino acid sequence of antigen 4 is the sequence SEQ NO:2 (44K) in the sequence listing.

[0049] 2. Antigen recombination expression

[0050] Codon optimization was performed on the gene sequences of four antigens according to the E. coli expression system.

[0051] The optimized gene sequence is as follows:

[0052] The gene sequence of antigen 1 is the sequence listed in SEQ NO:5;

[0053] The gene sequence of antigen 2 is the sequence listed in SEQ NO:6;

[0054] The gene sequence of antigen 3 is the sequence listed in SEQ NO:7.

[0055] The gene sequence of antigen 4 is the sequence listed in SEQ NO:8.

[0056] The four antigen gene sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. and ligated into the prokaryotic expression vector pET-30a to successfully construct a recombinant expression plasmid. The recombinant expression plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and after IPTG-induced expression, Ni-NTA affinity chromatography purification, dialysis and other processes, four recombinant antigen proteins were obtained and lyophilized for preservation. Figure 5The image shows SDS-PAGE images of four recombinant antigens, where 1 represents antigen 1, 2 represents antigen 2, 3 represents antigen 3, and 4 represents antigen 4.

[0057] 3. Antigen immunogenicity and reactivity screening

[0058] Four recombinant antigen proteins were reconstituted in 0.01 mol / L PBS (pH 7.2) at 4°C, and the concentration was adjusted to 100 μg / mL. Sodium chloride was then added to bring the concentration to 8.5 g / L. A 1 mol / L CaCl2 solution was slowly added dropwise to the antigen solution until a final concentration of 5 mmol / L was achieved, while stirring with a magnetic stirrer. After the addition was complete, stirring continued for 30 min to ensure complete ionic bond formation. Then, aluminum hydroxide suspension was slowly added dropwise, with stirring continued for 1 h to ensure the antigen was coated with CaCl2. 2+ Synergistic adsorption with aluminum hydroxide, followed by the addition of 0.01% polysorbate, and sterilization by filtration through a 0.22μm PES filter membrane, yielded four types of spray vaccines.

[0059] Adult sheep were immunized with four types of spray vaccines. 0.3 mL of the vaccine was sprayed into the nasal cavity. Two weeks after the first immunization, the sheep were immunized again. Two weeks after the second immunization, blood was collected to obtain four types of serum.

[0060] ELISA method establishment and detection:

[0061] a. After rejuvenating the PG3 strain of mycoplasma in adult sheep, it was cultured in large quantities using mycoplasma culture medium. The mycoplasma in the logarithmic growth phase was ultrasonically fragmented to obtain the whole-cell antigen.

[0062] b. Dilute the whole cell antigen to 2 mg / mL with 0.05 mol / L, pH 9.6 carbonate buffer (coating solution), and prepare 100 μL / well in a 96-well microplate. Incubate at 4°C for 16 hours.

[0063] c. Shake off the liquid in the wells and wash three times with 300 μL of 0.05 mol / L carbonate buffer solution at pH 9.6.

[0064] d. Block with 0.05 mol / L carbonate buffer containing 10% BSA at pH 9.6 for 1 hour at 37°C.

[0065] e. After spin-drying, dry in a vacuum drying oven at 30°C for 1 hour.

[0066] f. Dilute the four serum samples and positive and negative serum standards 20-fold with PBST buffer containing 2% BSA at 0.02 mol / L and pH 7.2.

[0067] g. Add 100 μL of diluted serum to each well and incubate at 37°C for 1 hour.

[0068] h. Shake off the liquid in the wells and wash 5 times with 300 μL of 0.02 mol / L PBST buffer at pH 7.2.

[0069] i. Dilute horseradish peroxidase-labeled rabbit anti-sheep IgG 5000 times with PBST buffer containing 2% BSA at 0.02 mol / L and pH 7.2, and add 100 μL per well to the microplate. Incubate at 37°C for 1 hour.

[0070] j. Shake off the liquid in the wells and wash 5 times with 300 μL of 0.02 mol / L PBST buffer at pH 7.2.

[0071] k. Add 100 μL of single-component TMB colorimetric solution to each well and incubate at 37°C for 10 minutes.

[0072] 1. Add 100 μL of 2M sulfuric acid to each well to stop the reaction. Detect OD450 using an ELISA reader. Perform two parallel experiments for each sample.

[0073] The OD450 of the antibody in serum corresponding to each antigen is:

[0074] Antigen 1: 1.332, 1.278; Antigen 2: 1.952, 2.261; Antigen 3: 1.648, 1.992; Antigen 4: 1.331, 1.317; Positive serum: 1.352, 1.268; Negative serum: 0.072, 0.087.

[0075] Figure 6 The graph shows the reaction results of antibodies against four recombinant antigens. From top to bottom, the antigens are antigen 1, antigen 2, antigen 3, antigen 4, positive control, and negative control. The results indicate that all four antigens exhibit high immunogenicity and reactivity.

[0076] 4. Screening for the blocking effect of immune serum on MCCP-infected GTC cells

[0077] Negative serum, positive serum from MCCP-infected goats, and positive serum prepared from four antigens obtained in antigen immunogenicity and reactivity screening were mixed at a ratio of 1:10 with 1.5 × 10⁻⁶ serum. 7 After mixing with MCCP of copies / L, incubate at 37°C for 1 hour, then add to a 96-well cell culture plate containing well-grown GTC cells with a confluence of 90%, and react with the GTC cells. At the same time, untreated MCCP is set up as a normal control.

[0078] Two hours later, the unattached MCCP cells in the culture wells were washed away with PBS preheated to 37°C. The cells were then fixed with 40 g / L paraformaldehyde at room temperature for 30 min, followed by washing three times with PBS preheated to 4°C for 5 min each time. Next, 250 μL of 100 g / L skim milk powder solution preheated to 4°C was added to each well at 37°C for 30 min. The blocking solution was discarded, excess solution was removed, and 250 μL / well of MCCP-positive serum diluted 100 times with the blocking solution was added. The cells were incubated at 37°C in a 5% CO2 incubator for 1.5 h. Healthy goat serum was used as a negative control, and primary antibody against uninfected MCCP cells served as a blank control. After incubation, the cells were washed three times with PBS preheated to 4°C for 5 min each time. Under light-protected conditions, 250 μL / well of secondary antibody (FITC-rabbit anti-goat IgG antibody) diluted 5000 times with the blocking solution was added. The cells were then placed in a humidified chamber and incubated at 37°C in a 5% CO2 incubator for 1 h in the dark. Wash three times with PBS pre-cooled to 4°C for 5 minutes each time, and observe and photograph using an inverted microscope.

[0079] The result is Figure 7 The indirect immunofluorescence results are shown in the figure. A: Positive control – untreated MCCP, primary antibody is MCCP-positive serum; B: Negative control – untreated MCCP, primary antibody is MCCP-negative serum; C: Blank control – uninfected MCCP, primary antibody is MCCP-positive serum; D: MCCP treated with antigen 1 immune serum for blocking, primary antibody is MCCP-positive serum; E: MCCP treated with antigen 2 immune serum for blocking, primary antibody is MCCP-positive serum; F: MCCP treated with antigen 3 immune serum for blocking, primary antibody is MCCP-positive serum; G: MCCP treated with antigen 4 immune serum for blocking, primary antibody is MCCP-positive serum; H: Negative serum blocking – MCCP treated with negative serum for blocking, primary antibody is MCCP-positive serum.

[0080] The results show that antigen 1 and antigen 4 are the optimal antigens. Antigen 1 and antigen 4 are combined to form an antigen protein combination, with antigen 1 as the first antigen protein (SEQ NO:1 sequence in the sequence listing) and antigen 4 as the second antigen protein (SEQ NO:2 sequence in the sequence listing).

[0081] The mass ratio of the first antigen protein to the second antigen protein is 1:(1-1.5). Example 2

[0082] A nasal spray subunit vaccine for infectious pleuropneumonia in sheep, comprising a first solution, a second solution, and a third solution; wherein the volume ratio of the first solution, the second solution, and the third solution is 3:1:1.

[0083] The first solution comprises a 100 μg / mL combination of antigen proteins, wherein the mass ratio of the first antigen protein to the second antigen protein is 1:1.5 mmol / L calcium chloride, 0.01 mmol / mL aluminum hydroxide, 8.5 g / L sodium chloride, 0.01% polysorbate, and 0.01 mol / L PBS at pH 7.2 as the solvent.

[0084] The second solution comprises 0.8 mg / mL tannic acid, 80 μg / mL CpG ODN, 8.5 g / L sodium chloride, 0.005% disodium EDTA, and 0.01 mol / L PBS at pH 6.8 as the solvent.

[0085] The third solution comprises 2 mg / mL polydopamine, 8.5 g / L sodium chloride, and 0.01 mol / L PBS at pH 7.2 as a solvent.

[0086] In the first solution, polysorbate improves solubility and prevents component aggregation; in the second solution, disodium EDTA prevents metal ions from interfering with the stability of CpG ODN.

[0087] A method for preparing a nasal spray subunit vaccine against contagious pleuropneumonia in sheep:

[0088] First solution:

[0089] Antigen protein reconstitution: The freeze-dried first and second antigen proteins were reconstituted at 4°C in a 1:1 mass ratio with 0.01 mol / L PBS at pH 7.2. The concentration was adjusted to 100 μg / mL and then sodium chloride was added to bring the concentration to 8.5 g / L.

[0090] Ca 2+ Complex formation: At 4°C, slowly add 1 mol / L CaCl2 solution dropwise to the antigen solution until the final concentration is 5 mmol / L, stirring with a magnetic stirrer while adding. After the addition is complete, continue stirring for 30 min to ensure sufficient ionic bond formation. Then, slowly add aluminum hydroxide suspension dropwise, continuing stirring for 1 h to ensure the antigen is coated with CaCl2. 2+ The solution was obtained by synergistic adsorption with aluminum hydroxide, followed by the addition of 0.01% polysorbate and then sterilization by filtration through a 0.22μm PES filter membrane.

[0091] Second solution:

[0092] CpG ODN was dissolved in 0.01 mol / L sterile PBS solution at pH 6.8 to a final concentration of 80 μg / mL. Tannic acid powder was added to a final concentration of 0.8 mg / mL. The solution was vortexed at room temperature until completely dissolved. Sodium chloride was added to a final concentration of 8.5 g / L. Disodium EDTA (0.005% by mass) was added. The solution was then filtered through a 0.22 μm PES membrane for sterilization to obtain the second solution.

[0093] Third solution:

[0094] Polydopamine was added to 0.01 mol / L PBS at pH 7.2 to a final concentration of 2 mg / mL, and sodium chloride was added to a final concentration of 8.5 g / L. The solution was then sterilized by filtration through a 0.22 μm CA filter membrane to obtain the third solution.

[0095] When using this solution, spray the first, second, and third solutions into the sheep's nasal cavity in a volume ratio of 3:1:1.

[0096] Fluorescent PCR detection of vaccine protective efficacy

[0097] Vaccine sample setup: MCCP infection group; first solution vaccine immunization group; complete vaccine immunization group; negative control group.

[0098] Immunization dose:

[0099] The first solution vaccine immunization group was given 0.3 mL of the first solution sprayed into the nasal cavity; 0.1 mL of 8.5 g / L sodium chloride solution was given twice.

[0100] For the complete vaccine immunization group, 0.3 mL of the first solution was sprayed into the nasal cavity, 0.1 mL of the second solution was sprayed into the nasal cavity, and 0.1 mL of the third solution was sprayed into the nasal cavity.

[0101] The negative control group consisted of 0.1 mL of 8.5 g / L sodium chloride solution, repeated 3 times.

[0102] Experimental methods: Adult sheep in the above experimental groups were immunized. A second immunization was performed two weeks after the initial immunization. In the second week after the second immunization, the PG3 strain was administered at a dose of 1×10⁻⁶. 8 CFU / Infection was performed via nasal drops only. Nasal swabs were collected 5 days after infection for real-time quantitative PCR detection, and the animals' disease status was observed.

[0103] The probe used in real-time PCR is the sequence listed in SEQ NO:9.

[0104] Upstream primer F1 is the sequence of SEQ NO:10 in the sequence listing.

[0105] The downstream primer R1 is the sequence of SEQ NO:11 in the sequence listing.

[0106] Figure 8 The image shows the results of real-time quantitative PCR detection. In the image, 1 and 2 represent the MCCP infection group; 3 and 4 represent the first solution vaccine immunization group; 5 and 6 represent the complete vaccine immunization group; and 7 and 8 represent the negative control group.

[0107] The results show that:

[0108] The mean CT value of the MCCP infection group was 17.2, and clinical observation showed obvious symptoms such as unsteady standing, conjunctival congestion, frequent wet cough, and mucopurulent nasal discharge. Two experimental animals died 6 days and 9 days after challenge with the virus, respectively.

[0109] The first group immunized with the solution vaccine tested positive, with a mean CT value of 23.7. The animals exhibited lethargy, intermittent dry cough, and fever exceeding 40 degrees Celsius. One experimental animal died 10 days after challenge, while the other remained alive on day 28. Upon euthanasia, autopsy revealed significant fibrinous exudation and extensive dark red consolidation in the lungs.

[0110] The group that received the complete vaccine tested negative and showed no signs of illness during subsequent observation.

[0111] ELISA antibody monitoring

[0112] Vaccine sample setup: First solution vaccine immunization group (experimental group 1); complete vaccine immunization group (experimental group 2); adjuvant group (control group 1); blank group (control group 2).

[0113] Immunization dose:

[0114] The first solution vaccine immunization group was given 0.3 mL of the first solution sprayed into the nasal cavity; 0.1 mL of 8.5 g / L sodium chloride solution was given twice.

[0115] For the complete vaccine immunization group, 0.3 mL of the first solution was sprayed into the nasal cavity, 0.1 mL of the second solution was sprayed into the nasal cavity, and 0.1 mL of the third solution was sprayed into the nasal cavity.

[0116] The adjuvant group consists of the antigen protein combination removed from the first solution vaccine immunization group, and is otherwise the same as the first solution vaccine immunization group.

[0117] Experimental methods: Adult sheep in each of the above experimental groups were immunized. Serum was collected on the day of the first immunization. Two weeks later, a second immunization was performed and serum was collected again. Serum was then collected in the second week, the first month, the third month, and the sixth month after the second immunization. A third immunization was performed in the sixth month. Finally, a last serum collection was performed six months after the third immunization.

[0118] The ELISA detection method described in Example 1 was used to detect antibodies in the serum of each node. The results are shown in [Figure 1]. Figure 9 Table 1 shows the antibody levels at different time points after immunization with different component vaccines.

[0119] The results showed that administering a second immunization 2 weeks after the initial immunization and a booster immunization 6 months after the second immunization could maintain a high antibody titer for a long time and achieve a good protective effect.

[0120] Table 1. Antibody values ​​at different time points after immunization with different vaccine components

[0121]

[0122] The principle of the nasal spray ovine infectious pleuropneumonia subunit vaccine of this invention is as follows:

[0123] First, antigen stability optimization: Ca 2+ As an electrolyte, it can stabilize the secondary and tertiary structures of antigenic proteins through ionic bonds, and is especially suitable for protein antigens, reducing their degradation in nasal mucosal secretions and promoting absorption.

[0124] Second, optimization of adjuvant effect: tannic acid and Ca 2+ When the antigen complex binds, it causes the antigen to become particulate. Through the synergistic effect of polyphenols, metal ions, and proteins, a more ordered immunogenic complex network structure is formed, which slows down the rate at which the antigen is washed away or degraded by nasal secretions, prolongs the residence time of the antigen on the mucosa, and enhances the uptake and processing efficiency of antigen-presenting cells. In addition, tannic acid, as a polyphenol compound, can help enhance the vaccine-induced immune response by regulating the signaling pathways of immune cells (such as activating inflammation-related pathways such as NF-κB, or promoting cytokine secretion).

[0125] Third, enhanced mucosal adhesion: In the final step, PDA encapsulates the complex and utilizes its biomimetic adhesion properties, similar to mussel foot silk protein, to form a three-dimensional structure that firmly adheres to the nasal mucosa in a moist environment, prolonging antigen exposure time and promoting the continuous production of antibodies.

[0126] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An antigenic protein combination of Mycoplasma caprineis subspecies of caprine pneumonia, characterized by: Including the first antigen protein and the second antigen protein, The amino acid sequence of the first antigen protein is shown in SEQ NO:1, and the amino acid sequence of the second antigen protein is shown in SEQ NO:

2.

2. The antigen protein combination of Mycoplasma caprineis subspecies of caprine pneumonia according to claim 1, characterized in that: The mass ratio of the first antigen protein to the second antigen protein is 1:(1-1.5).

3. A method for preparing a nasal spray subunit vaccine against contagious pleuropneumonia in sheep, characterized in that, The preparation steps include: (1) Preparation of the first solution: Antigen protein combination reconstitution: The freeze-dried first antigen protein and second antigen protein according to any one of claims 1-2 are reconstituted in PBS at a mass ratio of 1:1 (pH 7.2, 0.01 mol / L) at 4°C, the concentration is adjusted to 100 μg / mL, and then sodium chloride is added to bring the concentration to 8.5 g / L. Ca²⁺ complex formation: At 4°C, 1 mol / L CaCl₂ solution was slowly added dropwise to the antigen-protein combination solution until the final concentration was 5 mmol / L. The mixture was stirred with a magnetic stirrer while adding the solution. After the addition was completed, the mixture was stirred for 30 min to ensure that the ionic bonds were fully formed. Then, aluminum hydroxide suspension was slowly added dropwise and stirred for 1 h to ensure that the antigen was synergistically adsorbed by Ca²⁺ and aluminum hydroxide. After adding 0.01% polysorbate, the first solution was obtained. (2) Preparation of the second solution: CpG ODN was dissolved in 0.01 mol / L sterile PBS solution at pH 6.8 to a final concentration of 80 μg / mL. Tannic acid powder was added to a final concentration of 0.8 mg / mL. The solution was vortexed at room temperature until completely dissolved. Sodium chloride was added to a final concentration of 8.5 g / L. Disodium EDTA (0.005% by mass) was then added to obtain the second solution. (3) Preparation of the third solution: Polydopamine was added to 0.01 mol / L PBS at pH 7.2-7.4 to a final concentration of 1-2 mg / mL, and sodium chloride was added to a final concentration of 8.5 g / L to obtain the third solution.

4. The method for preparing the nasal spray ovine infectious pleuropneumonia subunit vaccine according to claim 3, characterized in that: The first and second solutions are sterilized by filtration through a 0.22 μm PES filter membrane; the third solution is sterilized by filtration through a 0.22 μm CA filter membrane.

5. A nasal spray ovine infectious pleuropneumonia subunit vaccine prepared by the preparation method described in claim 3, characterized in that: Including the first solution, the second solution, and the third solution; The first solution contains the antigen protein combination and Ca²⁺; The second solution contains tannic acid; The third solution contains polydopamine.

6. The nasal spray ovine infectious pleuropneumonia subunit vaccine according to claim 5, characterized in that: The first solution comprises 50-100 μg / mL of antigen protein combination, 5-10 mmol / L of calcium chloride, 0.01 mmol / mL of aluminum hydroxide, 8.5 g / L of sodium chloride, and 0.01% by mass of polysorbate, using 0.01 mol / L PBS at pH 7.2-7.4 as the solvent.

7. The nasal spray ovine infectious pleuropneumonia subunit vaccine according to claim 5, characterized in that: The second solution comprises 0.5-1 mg / mL tannic acid, 80 μg / mL CpG ODN, 8.5 g / L sodium chloride, and 0.005% disodium EDTA, using 0.01 mol / L PBS at pH 6.5-7.0 as the solvent.

8. The nasal spray ovine infectious pleuropneumonia subunit vaccine according to claim 5, characterized in that: The third solution comprises 1-2 mg / mL of polydopamine and 8.5 g / L of sodium chloride, using 0.01 mol / L PBS at pH 7.2-7.4 as a solvent.

9. The nasal spray ovine infectious pleuropneumonia subunit vaccine according to claim 5, characterized in that: The first, second, and third solutions were sprayed into the nasal cavity of sheep in a volume ratio of 3:1:1.

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