Preparation method and application of pasteurization-bordetella nanometer vaccine

Abstract

The invention discloses a preparation method and application of a pasteurization-bordetella nanometer vaccine, and belongs to the technical field of biological medicine. Comprising the following steps: carrying out high-speed shearing emulsification on epigallocatechin gallate, an antigen water phase and soybean oil, and homogenizing to form a stable oil-in-water emulsion, namely the pasteurization-bordetella nano vaccine. A challenge test proves that the protection rate of the ESO vaccine to pasteurella multocida and bordetella bronchiseptica reaches up to 90%, which is obviously superior to the 40-70% protection rate of an aluminum adjuvant group, and meanwhile, the lung bacterium loading amount is obviously reduced, so that the ESO vaccine shows an excellent clinical protection effect. The EGCG and the vegetable oil are innovatively compounded, so that the limitation of the traditional adjuvant in the aspects of immune efficacy, safety and function singleness is broken through, and an efficient and safe novel vaccine solution is provided for prevention and control of pasteurellosis and bordetella disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing and applying a Pasteurella-Bordetella nanovaccine. Background Technology

[0002] Pasteurella multocida infection and bortezomib infection are two bacterial infectious diseases that seriously threaten the development of my country's aquaculture industry. They are caused by Pasteurella multocida (… Pasteurella multocida Pm) and Bordetella bronchiseptica ( Bordetella bronchiseptica The former is characterized by respiratory symptoms and septicemia, while the latter manifests as a chronic respiratory infection. Both are characterized by high morbidity and mortality rates, causing huge economic losses to the livestock industry. Scientific feeding management, strict quarantine procedures for introduced breeds, and immunization with a bivalent inactivated vaccine against Pasteurella and Bordetella are key measures to effectively control the above-mentioned diseases, ensure the health of livestock, and promote the stable development of the livestock industry.

[0003] Vaccine adjuvants, as a core component of vaccine formulations, can significantly enhance antigen immunogenicity, regulate immune response types, and prolong immune memory. However, currently used traditional vaccine adjuvants (such as aluminum and mineral oil) have significant limitations: aluminum adjuvants mainly induce Th2 humoral immunity, with insufficient stimulation of cellular immunity; while mineral oil adjuvants can enhance cellular immunity, they cause strong local inflammatory reactions and have poor biodegradability. Therefore, developing safe and efficient novel adjuvants has become an important direction in veterinary vaccine research. Vegetable oil emulsion adjuvants demonstrate significant advantages in this regard: vegetable oils (such as injectable soybean oil, meeting the standards of the Chinese Pharmacopoeia) have good biocompatibility and biodegradability, and can significantly reduce inflammatory reactions at the injection site; as a hydrophobic carrier, they can form stable oil-in-water (O / W) emulsions with antigens and immune-enhancing components through emulsification processes, not only achieving sustained-release delivery of antigens but also helping to stimulate the body to produce stronger humoral and cellular immune responses.

[0004] Epigallocatechin gallate (Epigallocatechin gallate) Epigallocatechin gallate EGCG (enzyme-enhanced iodine) is the main active polyphenol component in green tea, possessing various biological activities such as antioxidant, anti-inflammatory, antiviral, antitumor, and immune-enhancing effects. It can activate the function of immune cells such as macrophages and T cells. These properties give EGCG potential application value in activating the body's immune system and enhancing the effectiveness of vaccines. However, there are currently no reports on the preparation of novel adjuvants by combining EGCG with soybean oil. Summary of the Invention

[0005] The application aims to provide a preparation method and application of a Pasteurella-Bordetella nanovaccine to solve the problems of the prior art.

[0006] To achieve the above-mentioned purpose, the application provides the following solutions. One of the technical solutions of the application is a preparation method of a Pasteurella-Bordetella nanovaccine, which comprises the following steps: subjecting epigallocatechin gallate (EGCG), an antigen aqueous phase and soybean oil to high-speed shearing emulsification, and then subjecting the emulsion to homogenization to form a stable oil-in-water emulsion, which is the Pasteurella-Bordetella nanovaccine.

[0007] The Pasteurella-Bordetella nanovaccine prepared by the preparation method.

[0008] The Pasteurella-Bordetella nanovaccine is used for preparing a medicine for preventing and / or treating Pasteurella disease and / or Bordetella disease.

[0009] The application also provides a medicine for preventing and / or treating Pasteurella disease and / or Bordetella disease, which comprises the Pasteurella-Bordetella nanovaccine.

[0010] Based on the above technical solutions, the application has the following technical effects. The application forms a novel adjuvant ESO by compounding EGCG and soybean oil, and prepares the Pasteurella-Bordetella nanovaccine, which has the following remarkable beneficial effects. 1. The immunization effect is remarkable: the specific IgG antibody level induced by the ESO adjuvant is maintained at a high level, and the IgG1 and IgG2a subtype levels are both significantly higher than those of the traditional aluminum adjuvant.

[0011] 2. Multiple biological activities are synergistic: EGCG not only has an adjuvant effect, but also has antioxidant and anti-inflammatory properties, which can simultaneously improve the serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities during the immune process, significantly reduce the content of malondialdehyde (MDA), effectively reduce the oxidative stress damage related to immunity and infection, and form a synergistic effect of immune enhancement and antioxidant protection.

[0012] 3. Excellent protection effect: the challenge test proves that the protection rate of the ESO vaccine against Pasteurella multocida and Bordetella bronchiseptica is as high as 90%, which is significantly better than the protection rate of 40%-70% of the aluminum adjuvant group, and the lung bacterial load is significantly reduced, which shows excellent clinical protection effect.

[0013] 4. Good physicochemical properties: the oil-in-water (O / W) emulsion prepared by high-pressure homogenization process has uniform particle size (about 177 nm), stable Zeta potential (-40.97 mV), and good dispersibility, meeting the stability standard of the veterinary pharmacopoeia.

[0014] In summary, the present application breaks through the limitations of traditional adjuvants in immunization efficacy, safety, and single function by innovative compounding of EGCG and vegetable oil, and provides an efficient and safe new vaccine solution for the prevention and control of pasteurellosis and bovine granuloma disease. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For the physicochemical properties of the ESO-Pm-Bb vaccine emulsion. Among them, A: ESO- Pm - Bb vaccine emulsion dosage form; B: ESO- Pm - Bb vaccine emulsion particle size distribution chart; C: ESO- Pm - Bb vaccine emulsion stability; D: ESO- Pm - Bb vaccine emulsion TEM electron microscope chart (200 nm scale).

[0016] Figure 2 For the influence of ESO-Pm-Bb on the levels of specific antibody IgG and its subtypes. Among them, A: Pm IgG, Bb IgG levels in serum 1-6W after the second immunization; B: Pm IgG1, Bb IgG1, Pm IgG2a, Bb IgG2a levels in serum at the time of the second immunization 4W.

[0017] Figure 3 For the detection results of mouse spleen lymphocyte stimulation index SI and spleen T lymphocyte subpopulation CD4 + / CD8 + ratio. Among them, A: the proliferation of spleen lymphocytes of immunized mice under the stimulation of LPS / ConA / Pm-Bb was detected by CCK-8 method; B: the CD4 + / CD8 + ratio in (C) was counted; C: the proportion of CD4 + or CD8 + cells was detected by flow cytometry.

[0018] Figure 4 For the influence of ESO on the levels of cytokines in serum and lymphocyte supernatant. Among them, A: IFN-γ; IL-6; IL-10; IL-17 content in serum; B: IFN-γ; IL-6; IL-10; IL-17 content in cell supernatant.

[0019] Figure 5 Figure 9 is the influence of ESO-Pm-Bb on the survival rate and lung bacterial load of the challenged mice. Wherein, A: survival curve within 7 days after Pm challenge; B: survival curve within 7 days after Bb challenge; C: lung Pm bacterial load after 7 days of challenge; D: lung Bb bacterial load after 7 days of challenge.

[0020] Figure 6 Figure 10 is the influence of ESO-Pm-Bb on the antioxidant indexes in the serum and liver tissue supernatant of the mice. Wherein, A: serum antioxidant indexes after 28 days of double immunization; B: serum antioxidant indexes after 7 days of Pm challenge; C: serum antioxidant indexes after 7 days of Bb challenge. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the technical solutions described in the present application are conventional solutions in the art, and the reagents or raw materials used are purchased from commercial channels or are already disclosed.

[0022] The present application provides a preparation method of Pasteurella-Bordetella nanovaccine, comprising the following steps: subjecting epigallocatechin gallate, an antigen aqueous phase and soybean oil to high-speed shearing emulsification, and then subjecting to homogenization to form a stable oil-in-water emulsion, which is the Pasteurella-Bordetella nanovaccine.

[0023] In some specific embodiments, the volume ratio of the antigen aqueous phase and the soybean oil is 1:1, and the content of the epigallocatechin gallate is 5 ug / mL.

[0024] In some specific embodiments, the preparation method of the antigen aqueous phase is as follows: subjecting Pasteurella multocida and Bordetella bronchiseptica to recovery and expansion culture, resuspending with PBS after inactivation to obtain the antigen aqueous phase.

[0025] In some specific embodiments, the Pasteurella multocida is P. multocida (CVCC 500) strain, which is purchased from the National Veterinary Microbial Culture Collection Center; and the Bordetella bronchiseptica is Bb FX-1.

[0026] In some specific embodiments, the high-speed shearing emulsification time is 0.5-3 min.

[0027] In some specific embodiments, the homogenization pressure is 800-1200 pa, and the homogenization is performed for 2-10 cycles.

[0028] The present application also provides the Pasteurella-Bordetella nanovaccine prepared by the preparation method.

[0029] The application also provides application of the Pasteurella-Bovis nanovaccine in preparation of a medicine for preventing and / or treating pasteurellosis and / or bovis disease.

[0030] The application also provides a medicine for preventing and / or treating pasteurellosis and / or bovis disease, which comprises the Pasteurella-Bovis nanovaccine.

[0031] Embodiment 1 1. Materials and methods 1.1 Test animals and main reagents SPF grade ICR female mice 104 (20 ± 2 g) were purchased from Hangzhou Hengsi Biological Technology Co., Ltd., and were raised in the animal center of Zhejiang Academy of Agricultural Sciences, and were fed with sterile feed and water, the environmental temperature was (25 ± 1) ℃, the humidity was (55 ± 5) %, and the formal test was started after pre-feeding for 1 week.

[0032] Soybean oil (SO) was purchased from Zhejiang Tianyushan Pharmaceutical Co., Ltd., which met the standard of injection oil in Chinese Pharmacopoeia; epigallocatechin gallate (EGCG) was purchased from Xi'an Ruining Biological Technology Co., Ltd., with a purity of 98%.P. multocida The CVCC500 strain was derived from a strain purchased from the National Veterinary Microbial Culture Collection Center; the Bb FX-1 strain was derived from a strain preserved in the Livestock Bacterial Disease Research Room of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, which has been disclosed in the patent CN116983396A; tryptic soy agar (TSA), tryptone soyabroth (TSB) and Martin broth medium (MB) were purchased from Thermo Fisher Scientific Company, USA. Aluminum adjuvant was purchased from Xibao Biological Technology (Shanghai) Co., Ltd. HRP-labeled goat anti-mouse IgG, IgG1 and IgG2a antibodies were purchased from Abeam Company; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich Company; concanavalin A (ConA) was purchased from APExBIO Company, USA; CCK-8 was purchased from Hangzhou Luoke Biological Technology Co., Ltd. Penicillin (50 IU / mL), streptomycin (50 IU / mL), fetal bovine serum (FBS), RPMI-1640, flow cytometry anti-mouse antibodies (CD4+, CD8+ and the like) and cytokine INF-γ, IL-6, IL-10 and IL-17 ELISA kits were purchased from Lianke Biological Technology Co., Ltd.; mouse anti-oxidative T-AOC, SOD, GSH-PX and MDA kits were purchased from Nanjing Jiancheng Biological Engineering Company.

[0033] 1.2 Methods 1.2.1 Preparation of vaccine Recovery Pm, Bb FX-1 strain, pick single colony inoculated in Martin broth (Pm), TSB culture solution (Bb FX-1), 37℃, 200 r / min shaking culture for 12 h, then enlarge culture for 16-18 h at 1:100 ratio, count after coating plate, then inactivate with final concentration 0.27% formaldehyde for 18 h. Inactivated bacterial solution is washed with sterile normal saline for 4 times, finally resuspended with PBS to the required concentration, which is the antigen aqueous phase: Pm 2×10 9 CFU / mL, Bb FX-1 2×10 9 CFU / mL.

[0034] Mix the antigen aqueous phase with soybean oil SO oil phase (v:v=1:1), add EGCG (5 ug / mL), high-speed shear emulsification for 2 min (IKA T 10 basic type ultra-high speed homogenizer, ULTRA-TURRAX®), then pass through high-pressure homogenizer at 800 pa, 1000 pa, 1200 pa pressure for 5 cycles (ATS high-pressure homogenizer AH-1500), finally form stable oil-in-water (O / W) emulsion.

[0035] Determine the particle size distribution, polydispersity index (PDI) and Zeta potential of the emulsion by Nano ZS type nanoparticle size and Zeta potential analyzer. The type of emulsion is identified by dilution method, and the stability is determined according to the relevant provisions of Chinese Veterinary Pharmacopoeia (10 mL emulsion is centrifuged at 3000 r / min for 15 min, and the water phase ≤0.5 mL is considered qualified). After the type and stability of the vaccine emulsion are qualified, the ESO-Pm-Bb vaccine is obtained. The EGCG content of the vaccine is 5 ug / mL, and the final bacterial content of the vaccine is Pm 1×10 9 CFU / mL + Bb FX-1 1×10 9 CFU / mL.

[0036] The alum adjuvant vaccine is prepared according to the manufacturer's instructions, and the alum and antigen aqueous phase are mixed for 2 min, which is the Alum-Pm-Bb vaccine. The final bacterial content of the vaccine is Pm 1×10 9 CFU / mL + Bb FX-1 1×10 9 CFU / mL.

[0037] 1.2.2 Animal grouping and immunization program Select 6-8 week old ICR female mice, and randomly divide them into 4 groups, 26 in each group. The specific grouping is as follows: (1) Blank control group (PBS): inject PBS; (2) Antigen group (Pm-Bb): inject Pm-Bb antigen; (3) Alum group (Alum-Pm-Bb): Administered Alum-Pm-Bb vaccine; (4) ESO group (ESO-Pm-Bb): ESO-Pm-Bb vaccine was administered.

[0038] Mice in each experimental group were immunized twice via intramuscular injection with an interval of 2 weeks.

[0039] 1.2.3 Detection of serum-specific antibodies and IgG subtypes During weeks 1-6 post-secondary immunization, blood was collected from 6 mice in each group, and serum was separated. The levels of specific IgG antibodies and their IgG1 and IgG2a subtypes were determined using an indirect ELISA method. Pm and Bb proteins were used as coating antigens, diluted to a final concentration of 2 µg / mL with CBS carbonate buffer, and 100 µL was added to each well of a 96-well ELISA plate. The plates were incubated at 37°C for 2 h, then transferred to 4°C for overnight incubation. After washing with phosphate buffer containing 0.05% Tween-20 (PBST), the plate was sealed with 200 µL of 5% (w / v) skim milk powder and incubated at 37°C for 2 h, followed by washing. Then, 100 µL of the test serum (1:2000) was added to each well, and the plate was incubated at 37°C for 1 h, followed by washing. Next, 100 µL of horseradish peroxidase-labeled goat anti-mouse lgG / lgG1 / lgG2a (1:10000) was added to each well, and the plate was incubated at 37°C for 1 h, followed by washing. Then, 100 µL of TMB substrate chromogenic solution was added to each well, and the reaction was carried out at 37°C in the dark for 15 min. Finally, 100 µL of 2MH2SO4 stop solution was added to each well to terminate the reaction, and the absorbance (OD) at 450 nm was measured using a microplate reader. 450nm ).

[0040] 1.2.4 Detection of splenic lymphocyte proliferation and T lymphocyte subsets Twenty-eight days after the second immunization, six mice from each group were sacrificed, and their spleens were aseptically collected. The spleens were ground and filtered through a 200-mesh cell sieve to prepare a single-cell suspension. Red blood cells were removed using erythrocyte lysis buffer, and the cells were resuspended in RPMI-1640 complete medium (containing 10% FBS and 1% penicillin-dextrose antibiotics) and the concentration was adjusted to 5 × 10⁻⁶ cells / mL. 6 Cells / mL. 100 μL / well of cell suspension was seeded into 96-well culture plates, and 100 μL of 1 μg / mL ConA, 5 μg / mL LPS, and 1×10⁻⁶ cells / mL were added respectively. 9CFU / mL inactivated Pm-Bb was used to stimulate mouse lymphocytes in each group, and cell control wells were set up with only RPMI 1640 complete medium. After culturing in a 37℃, 5% CO2 incubator for 44 h, 20 μL of CCK-8 solution was added to each well in the dark, and the cells were cultured for another 2 h. The absorbance (OD) value was then measured at 450 nm using a microplate reader. 450nm Calculate the lymphocyte stimulation index (SI) for each group using the following formula: SI = (Stimulation well OD) / (Stimulation well OD) 450nm - Blank Hole OD 450nm ) / (Unstimulated pore OD 450nm - Blank Hole OD 450nm ).

[0041] Separately, spleen lymphocyte suspension was taken and anti-mouse CD4 was added. + and CD8 + After gentle mixing with the flow cytometry antibody, incubate at room temperature in the dark for 30 min. Centrifuge at 1500 r / min for 8 min, discard the supernatant, resuspend cells in PBS, and analyze using a FACS Canto™ flow cytometer. Analyze CD4 using FlowJo software (version 10.0). + and CD8 + T lymphocyte percentage and CD4 count + / CD8 + ratio.

[0042] 1.2.5 Cytokine Level Detection Twenty-eight days after the second immunization, mouse serum was collected; simultaneously, splenic lymphocytes prepared in 1.2.4 were used to extract 5 × 10⁻⁶ cells. 6 Lymphocytes were seeded at a concentration of 1 / mL in 6-well plates and stimulated with inactivated Pm-Bb antigen for 72 h. The supernatant was then collected. Following strict adherence to the ELISA kit instructions, the levels of IFN-γ, IL-6, IL-10, and IL-17 in serum and cell supernatant were measured.

[0043] 1.2.6 Challenge protection test and lung bacterial load detection Forty-two days after the second immunization, a challenge test was conducted. Each mouse in the Pm challenge group was intraperitoneally injected with 0.2 mL of Pm bacterial solution (dose 1×10⁻⁶). 2 CFU / mL), each mouse in the Bb FX-1 challenge group was intraperitoneally injected with 0.2 mL of Bb FX-1 bacterial solution (dose 5 × 10⁻⁶ CFU / mL). 8 (CFU / mL). Mice were observed continuously for 7 days, and the survival rate was recorded.

[0044] Seven days after challenge, four mice in each group were sacrificed, and lung tissue was aseptically harvested. The tissue homogenate was serially diluted, and an appropriate amount of the diluted solution was spread on TSA plates. After incubation at 37°C for 24-36 h, the number of colonies was counted, and the bacterial load (CFU / mL) in each mL of lung tissue homogenate was calculated.

[0045] 1.2.7 Antioxidant index detection Serum was collected from mice 28 days after the second immunization and 7 days after challenge. Following the kit instructions, the levels of MDA (TBA method), GSH-PX (colorimetric method), T-AOC (ABTS method), and SOD activity (NBT method) in the mouse serum were measured.

[0046] 2 Results 2.1 Identification of the physicochemical properties of ESO-Pm-Bb vaccine emulsion To optimize vaccine preparation processes, this invention compared the effects of different homogenization pressures (800 Pa, 1000 Pa, 1200 Pa) on emulsion properties. Through comprehensive evaluation of emulsion type, stability, particle size, zeta potential, and polydispersity index (PDI), the results showed that the emulsions under all preparation conditions were oil-in-water (O / W) emulsions, and their stability met the requirements of the Chinese Veterinary Pharmacopoeia (<0.5 mL of aqueous phase precipitated after centrifugation of 10 ml emulsion).

[0047] Table 1 Physicochemical properties of ESO-Pm-Bb vaccine emulsions prepared under different homogenization pressures

[0048] Note: Different letters following the data in the same row indicate significant differences. P <0.05), where the same letter indicates no significant difference (P>0.05). The same applies to the following table.

[0049] As shown in Table 1, as the homogenization pressure increased from 800 Pa to 1200 Pa, the absolute value of the Zeta potential of the emulsion increased significantly from (-31.97 ± 1.53) mV to (-40.97 ± 1.12) mV. In terms of particle size, the emulsion prepared under the 1200 Pa condition had the smallest average particle size (177.63 ± 4.94) nm, and its PDI value was (10.90% ± 2.26%), which was slightly higher than the other two groups, but still lower than 20%.

[0050] Based on comprehensive assessment, 1200 Pa is the optimal homogenization pressure. Under this condition, the emulsion appears uniformly milky white, and the dilution method confirms it as an oil-in-water (O / W) formulation. Figure 1 (A); its particle size distribution curve is a single sharp peak type ( Figure 1 (B) The aqueous phase precipitated after centrifugation of 10 ml emulsion was less than 0.5 mL.Figure 1 (C) Transmission electron microscopy (TEM) observation showed that the droplets were regularly shaped and evenly distributed. Figure 1 (D).

[0051] 2.2 Immunogenic effect of ESO on Pasteurella multocida-Bordetella bronchiseptica inactivated vaccine 2.2.1 Effects of ESO-Pm-Bb on the levels of Pm, Bb, and FX-1 specific antibodies and their subtypes in mouse serum To assess the enhancing effect of ESO adjuvant on humoral immunity, serum levels of specific antibody IgG were dynamically monitored over 6 weeks following the second immunization. Figure 2 As shown in Figure A, the levels of Pm and Bb FX-1 specific IgG antibodies in the ESO group were significantly higher than those in other groups at each time point after the second immunization. P <0.05. In the ESO group, Pm-specific IgG reached a high level one week after the second immunization and remained at that level for 6 weeks; Bb FX-1-specific IgG peaked 3-5 weeks after the second immunization, slightly decreased in week 6 but remained significantly higher than in the antigen group and the aluminum adjuvant group. P <0.05%. Analysis of antibody subtypes at 28 days after the peak antibody level following the second immunization showed ( Figure 2 In the ESO group, the levels of Pm-IgG1, Pm-IgG2a, Bb-IgG1, and Bb-IgG2a antibodies induced were significantly higher than those in the antigen group and the aluminum adjuvant group. P <0.05).

[0052] 2.2.2 Detection of spleen T lymphocyte subsets and lymphocyte proliferation assay Detection of spleen lymphocyte proliferation assay using CCK-8 assay Figure 3 (A). After LPS stimulation, there was no significant difference in the Stimulation Index (SI) between the vaccine groups and the control group. P >0.05); After stimulation with ConA and Pm-Bb antigens, the SI values ​​of ESO were significantly higher than those of the antigen group ( P <0.0001 and P <0.01. Splenic T lymphocyte subsets were analyzed by flow cytometry ( Figure 3 (B, C). The results showed that CD4+ was present in the spleen lymphocytes of the ESO group mice. + / CD8 + The T cell ratio was significantly higher in the antigen group and the aluminum gel group. P <0.05. There was no statistically significant difference between the Pm-Bb group and the Alum group ( P >0.05).

[0053] 2.2.3 Detection of cytokine levels in mouse serum and lymphocyte supernatant Serum cytokine results showed (Figure 4 In the ESO group (A), the levels of IFN-γ, IL-6, IL-10, and IL-17 were significantly higher than those in the PBS and antigen groups (P<0.01 or P<0.0001), with the levels of IFN-γ, IL-6, and IL-17 being significantly higher than those in the aluminum adjuvant group (P<0.001 or P<0.0001). Lymphocyte supernatant cytokine results showed ( Figure 4 In the B group, the levels of all four cytokines in the ESO group were significantly higher than those in the PBS group and the antigen group (P<0.01 to P<0.0001), and the levels of IFN-γ, IL-6, and IL-10 were significantly higher than those in the aluminum adjuvant group (P<0.01 or P<0.001).

[0054] 2.2.4 Effects of ESO-Pm-Bb on the protection rate and lung bacterial load in mice after challenge To verify the protective effect of the vaccine, mice in each group were intraperitoneally injected with 0.2 mL of Pm bacterial solution (2 × 10⁻⁶). 1 CFU / mL), Bb FX-1 bacterial liquid (5×10 8 A challenge test was conducted using CFU / mL. The results of the challenge test showed ( Figure 5 : 7 days after challenge with Pm, all mice in the control group developed severe respiratory symptoms (lethargy, dyspnea, cough), with a mortality rate of 100% and a survival rate of 0%. The survival rates in the Pm-Bb group were 20%, in the Alum group 40%, and in the ESO group 90%. Compared with the PBS and Pm-Bb groups, the survival rate in the ESO group was significantly higher. P <0.05).

[0055] A 7-day observation of mice challenged with Bb FX-1 revealed that: in the control group, all mice died within 3 days of challenge, with a survival rate of 0%; the survival rates in the Pm-Bb group were 60%, in the Alum group 70%, and in the ESO group 90%. Compared with the PBS and Pm-Bb groups, the survival rate of the ESO group was significantly higher. P <0.05).

[0056] The results of lung tissue bacterial load detection were consistent with the survival rate. Figure 5 The results showed that the bacterial load of Pm and Bb FX-1 in the lung tissue of ESO group mice was significantly lower than that in PBS control group, antigen group and ( P <0.05).

[0057] 2.2.5 Detection of serum antioxidant indicators before and after virus challenge To further explore the mechanism by which EGCG enhances the immune response, this study detected antioxidant markers in the serum of mice 28 days after secondary immunization and 7 days after challenge. Figure 6 ). 28 days after the second vaccination ( Figure 6Group A), 7 days after challenge with Pm (Group B) Figure 6 Group A), 7 days after challenge with Pm (Group B) Figure 6 Group C), the total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities of serum in ESO group were significantly higher than those in antigen group and aluminum adjuvant group (P<0.05) P <0.0001 or P <0.001). In contrast, the content of malondialdehyde (MDA) representing the degree of lipid peroxidation damage was significantly reduced in ESO group (P<0.05). P <0.05). This indicates that ESO adjuvant can effectively improve the overall antioxidant status of the body and reduce the oxidative stress damage that may occur during the immune and infection process.

[0058] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For ordinary skilled users in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments are not required to be exhausted, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the claims of the present application.

Claims

1. A method for preparing a Pasteurella-Bordetella nanovaccine, characterized in that, Includes the following steps: Epigallocatechin gallate, antigen aqueous phase and soybean oil are emulsified by high-speed shearing, and then homogenized to form a stable oil-in-water emulsion, which is the Pasteurella-Bordetella nanovaccine.

2. The preparation method according to claim 1, characterized in that, The volume ratio of the antigen aqueous phase to soybean oil is 1:1, and the content of epigallocatechin gallate is 5 ug / mL.

3. The preparation method according to claim 1, characterized in that, The preparation method of the antigen aqueous phase is as follows: Pasteurella multocida and Bordetella bronchiseptica are resuscitated, cultured on a large scale, inactivated, and resuspended in PBS to obtain the antigen aqueous phase.

4. The preparation method according to claim 3, characterized in that, The Pasteurella multocida species is P. multocida The strain (CVCC 500) was purchased from the National Veterinary Microbial Culture Collection Center; the Bordetella bronchiseptica strain was Bb FX-1.

5. The preparation method according to claim 1, characterized in that, The high-speed shear emulsification time is 0.5~3 min.

6. The preparation method according to claim 1, characterized in that, The homogenization pressure is 800~1200pa, and the homogenization is carried out for 2-10 cycles.

7. Pasteurella multocida nanovaccine prepared by the preparation method according to any one of claims 1-6.

8. The use of the Pasteurella-Botrytis cinerea nanovaccine as described in claim 7 in the preparation of medicaments for the prevention and / or treatment of Pasteurella multocida and / or Botrytis cinerea.

9. A medicine for the prevention and / or treatment of pasteurellosis and / or bortezomibosis, characterized in that, Includes the Pasteurella-Botrytis cinerea nanovaccine as described in claim 7.

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