Preparation method and application of a b. pasteurii nanovaccine

Abstract

This invention discloses a method for preparing and applying a Pasteurella-Bordetella nanovaccine, belonging to the field of biomedical technology. The method includes the following steps: emulsifying epigallocatechin gallate, an antigen aqueous phase, and soybean oil under high-speed shearing, followed by homogenization to form a stable oil-in-water emulsion, which is the Pasteurella-Bordetella nanovaccine. Challenge experiments confirmed that the ESO vaccine achieved a protection rate of up to 90% against Pasteurella multocida and Bordetella bronchiseptica, significantly superior to the 40%-70% protection rate of the aluminum adjuvant group. Simultaneously, it significantly reduced the bacterial load in the lungs, demonstrating excellent clinical protective effects. This invention, through the innovative combination of EGCG and vegetable oil, overcomes the limitations of traditional adjuvants in terms of immunogenicity, safety, and single function, providing a highly efficient and safe new vaccine solution for the prevention and control of Pasteurella multocida and Bordetella multocida infections.

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 eosinophils) is the main active polyphenol component in green tea, possessing various biological activities such as antioxidation, anti-inflammation, antiviral, antitumor, and immune enhancement. 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 purpose of this invention is to provide a method for preparing and applying Pasteurella multocida nanovaccines to address the problems existing in the prior art. This invention prepares a novel adjuvant ESO by compounding EGCG with soybean oil, and then emulsifies it with Pm-Bb antigen to prepare a vaccine. The physicochemical properties and immunomodulatory effects are systematically evaluated, aiming to provide a theoretical basis for the development of highly effective veterinary vaccine adjuvants.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of the present invention is a method for preparing Pasteurella-Bordetella nanovaccine, comprising the following steps: emulsifying epigallocatechin gallate, antigen aqueous phase and soybean oil by high-speed shearing, and then homogenizing to form a stable oil-in-water emulsion, which is the Pasteurella-Bordetella nanovaccine.

[0008] The second technical solution of the present invention is a Pasteurella-Bordetella nanovaccine prepared by the preparation method.

[0009] The third technical solution of the present invention is the application of the Pasteurella-Botrytis cinerea nanovaccine in the preparation of drugs for the prevention and / or treatment of Pasteurella multocida and / or Botrytis cinerea.

[0010] The fourth technical solution of the present invention is a drug for the prevention and / or treatment of Pasteurella multocida infection and / or Bordetella multocida infection, comprising the Pasteurella multocida-Bordetella nanovaccine.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] This invention combines epigallocatechin gallate (EGCG) with soybean oil to form a novel adjuvant ESO, and uses this to prepare Pasteurella multocida nano-vaccines, achieving the following significant beneficial effects:

[0013] 1. Significant immune-enhancing effect: The level of specific IgG antibodies induced by ESO adjuvant remained high, and the levels of IgG1 and IgG2a subtypes were significantly higher than those of traditional aluminum adjuvant.

[0014] 2. Synergistic effect of multiple bioactivities: EGCG not only exerts an adjuvant effect, but its own antioxidant and anti-inflammatory properties enable the vaccine to simultaneously enhance serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity during the immunization process, significantly reduce malondialdehyde (MDA) content, effectively alleviate immune and infection-related oxidative stress damage, and form a synergistic effect of immune enhancement and antioxidant protection.

[0015] 3. Excellent protective efficacy: Challenge trials have confirmed that the ESO vaccine has a protection rate of up to 90% against Pasteurella multocida and Bordetella bronchiseptica, which is significantly better than the 40%-70% protection rate of the aluminum adjuvant group. At the same time, the bacterial load in the lungs is significantly reduced, demonstrating excellent clinical protective effect.

[0016] 4. Excellent physicochemical properties: The water-in-oil (O / W) emulsion prepared by high-pressure homogenization process has uniform particle size (approximately 177 nm), stable zeta potential (-40.97 mV), and good dispersibility, meeting the stability standards of the Veterinary Pharmacopoeia.

[0017] In summary, this invention, through the innovative combination of EGCG and vegetable oil, overcomes the limitations of traditional adjuvants in terms of immunogenicity, safety, and single function, providing a highly efficient and safe new vaccine solution for the prevention and control of Pasteurella multocida and Bordetella multocida infections. Attached Figure Description

[0018] Figure 1 For the identification of the physicochemical properties of ESO-Pm-Bb vaccine emulsion. Among them, A: ESO- Pm - Bb Vaccine emulsion formulation; B: ESO- Pm - Bb Vaccine emulsion particle size distribution diagram; C: ESO- Pm - Bb Vaccine emulsion stability; D: ESO- Pm - Bb TEM image of vaccine emulsion (200nm scale bar).

[0019] Figure 2 The effect of ESO-Pm-Bb on the levels of specific antibody IgG and its subtypes. A: Serum levels of Pm IgG and Bb IgG 1-6 weeks after the second immunization; B: Serum levels of Pm IgG1, Bb IgG1, Pm IgG2a, and Bb IgG2a 4 weeks after the second immunization.

[0020] Figure 3 The mouse spleen lymphocyte stimulation index (SI) and spleen T lymphocyte subset CD4 + / CD8 + Proportional detection results. A: Detection of splenic lymphocyte proliferation in immunized mice under LPS / ConA / Pm-Bb stimulation using the CCK-8 assay; B: Statistical analysis of CD4+ in (C). + / CD8 + Ratio; C: CD4 detected by flow cytometry + or CD8 + Cell ratio.

[0021] Figure 4 The effects of ESO on cytokines in serum and lymphocyte supernatant. A: Serum levels of IFN-γ, IL-6, IL-10, and IL-17; B: Cell supernatant levels of IFN-γ, IL-6, IL-10, and IL-17.

[0022] Figure 5 The effects of ESO-Pm-Bb on the protection rate and lung bacterial load in challenged mice were investigated. A: Survival curve of Pm within 7 days after challenge; B: Survival curve of Bb within 7 days after challenge; C: Lung Pm bacterial load 7 days after challenge; D: Lung Bb bacterial load 7 days after challenge.

[0023] Figure 6 The effects of ESO-Pm-Bb on antioxidant markers in mouse serum and liver tissue supernatant were investigated. A: serum antioxidant markers 28 days after secondary immunization; B: serum antioxidant markers 7 days after Pm challenge; C: serum antioxidant markers 7 days after Bb challenge. Detailed Implementation

[0024] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0025] This invention provides a method for preparing Pasteurella-Bordetella nanovaccine, comprising the following steps: emulsifying epigallocatechin gallate, an aqueous phase of antigen, and soybean oil under high-speed shearing, and then homogenizing to form a stable oil-in-water emulsion, which is the Pasteurella-Bordetella nanovaccine.

[0026] In some specific implementations, the volume ratio of the antigen aqueous phase to soybean oil is 1:1, and the content of epigallocatechin gallate is 5 ug / mL.

[0027] In some specific implementations, the preparation method of the antigen aqueous phase is as follows: resuscitate Pasteurella multocida and Bordetella bronchiseptica, expand culture, inactivate and resuspend in PBS to obtain the antigen aqueous phase.

[0028] In some specific implementations, the Pasteurella multocida is P. multocida. multocida The strain (CVCC 500) was purchased from the National Veterinary Microbial Culture Collection Center; the Bordetella bronchiseptica strain was Bb FX-1.

[0029] In some specific implementations, the high-speed shear emulsification time is 0.5 to 3 minutes.

[0030] In some specific implementations, the homogenization pressure is 800~1200 Pa, and the homogenization is performed for 2-10 cycles.

[0031] This invention also provides Pasteurella-Bordetella nanovaccine prepared by the aforementioned preparation method.

[0032] The present invention also provides the use of the Pasteurella-Botrytis nanovaccine in the preparation of medicaments for the prevention and / or treatment of Pasteurella multocida and / or Botrytis cinerea.

[0033] This invention also provides a medicament for the prevention and / or treatment of Pasteurella multocida and / or Bordetella multocida, including the Pasteurella multocida-Bordetella nanovaccine.

[0034] Example 1

[0035] 1. Materials and Methods

[0036] 1.1 Experimental animals and main reagents

[0037] One hundred and four SPF-grade female ICR mice (20 ± 2 g) were purchased from Hangzhou Hangsi Biotechnology Co., Ltd. The animals were housed at the Animal Center of Zhejiang Academy of Agricultural Sciences, fed with sterile feed and water, at an ambient temperature of (25 ± 1)℃ and a humidity of (55 ± 5)%. The formal experiment began one week after pre-feeding.

[0038] Soybean oil (SO) was purchased from Zhejiang Tianyushan Pharmaceutical Co., Ltd., and met the standards for injectable oils in the Chinese Pharmacopoeia; epigallocatechin gallate (EGCG) was purchased from Xi'an Ruiying Biotechnology Co., Ltd., with a purity of 98%. multocidaThe (CVCC500) strain was purchased from the National Veterinary Microbial Culture Collection Center; the Bb FX-1 strain was preserved in the Animal Bacterial Diseases Research Laboratory of the Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, and this strain has been disclosed in patent CN116983396A; Tryptic soy agar (TSA), Tryptone soybroth (TSB), and Martin broth (MB) were purchased from Thermo Fisher Scientific, Inc., USA. Aluminum adjuvant was purchased from Xibao Biotechnology (Shanghai) Co., Ltd. HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a antibodies were purchased from Abcam; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich; concanavalin A (ConA) was purchased from APExBIO, Inc., USA; and CCK-8 was purchased from Hangzhou Luoke Biotechnology Co., Ltd. ELISA kits for penicillin (50 IU / mL), streptomycin (50 IU / mL), fetal bovine serum (FBS), RPMI-1640, flow cytometry anti-mouse antibodies (CD4+, CD8+, etc.), and cytokines INF-γ, IL-6, IL-10, and IL-17 were purchased from Linko Biotechnology Co., Ltd.; mouse antioxidant T-AOC, SOD, GSH-PX, and MDA kits were purchased from Nanjing Jiancheng Bioengineering Co., Ltd.

[0039] 1.2 Methods

[0040] 1.2.1 Vaccine preparation

[0041] Resuscitate *Pm* and *Bb* FX-1 strains, and inoculate single colonies into Martin broth (*Pm*) and TSB medium (*Bb* FX-1*), respectively. Incubate at 37°C with shaking at 200 rpm for 12 h, then scale up the culture at a 1:100 ratio for 16–18 h. After plating and counting, inactivate the bacteria with 0.27% formaldehyde for 18 h. Wash the inactivated bacterial solution four times with sterile physiological saline, and finally resuspend in PBS to the required concentration to obtain the antigen aqueous phase: *Pm* 2 × 10⁻⁶. 9 CFU / mL, Bb FX-1 2×10 9 CFU / mL.

[0042] The antigen aqueous phase was mixed with the soybean oil SO phase (v:v=1:1), EGCG (5 ug / mL) was added, and the mixture was emulsified by high-speed shearing for 2 min (IKA T 10 basic type ultra-high-speed homogenizer, ULTRA-TURRAX®). Then, it was homogenized for 5 cycles at pressures of 800 Pa, 1000 Pa, and 1200 Pa (ATS high-pressure homogenizer AH-1500) to finally form a stable oil-in-water (O / W) emulsion.

[0043] The Nano ZS type nano particle size and Zeta potential analyzer was used to determine the particle size distribution, polydispersity index (PDI) and Zeta potential of the emulsion. The emulsion type was identified by the dilution method, and the stability was determined according to the relevant regulations of the Chinese Veterinary Pharmacopoeia (it was regarded as qualified if the volume of the separated aqueous phase was ≤ 0.5 mL after centrifuging 10 mL of the emulsion at 3000 r / min for 15 min). After the emulsion type and stability of the vaccine were both qualified, the ESO-Pm-Bb vaccine was obtained. The content of EGCG in the vaccine was 5 ug / mL, and the final bacterial content of the vaccine was Pm 1×10 9 CFU / mL + Bb FX-1 1×10 9 CFU / mL.

[0044] The Alum adjuvant vaccine was prepared according to the manufacturer's instructions. The Alum and the antigen aqueous phase were mixed for 2 min, that is, the Alum-Pm-Bb vaccine. The final bacterial content of the vaccine was Pm 1×10 9 CFU / mL + Bb FX-1 1×10 9 CFU / mL.

[0045] 1.2.2 Animal grouping and immunization procedure

[0046] ICR female mice at 6 - 8 weeks of age were selected and randomly divided into 4 groups, with 26 mice in each group. The specific grouping was as follows:

[0047] (1) Blank control group (PBS): Injected with PBS;

[0048] (2) Antigen group (Pm-Bb): Injected with Pm-Bb antigen;

[0049] (3) Alum group (Alum-Pm-Bb): Injected with Alum-Pm-Bb vaccine;

[0050] (4) ESO group (ESO-Pm-Bb): Injected with ESO-Pm-Bb vaccine.

[0051] Each experimental group of mice was intramuscularly injected with 0.2 mL, immunized 2 times, with an interval of 2 weeks.

[0052] 1.2.3 Detection of serum specific antibodies and IgG subtypes

[0053] 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 ).

[0054] 1.2.4 Detection of splenic lymphocyte proliferation and T lymphocyte subsets

[0055] 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. 9 CFU / 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 = (OD of stimulation well) / (OD of stimulation well) 450nm - Blank Hole OD 450nm ) / (Unstimulated pore OD 450nm - Blank Hole OD 450nm ).

[0056] 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.

[0057] 1.2.5 Cytokine Level Detection

[0058] 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.

[0059] 1.2.6 Challenge protection test and lung bacterial load detection

[0060] 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.

[0061] 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.

[0062] 1.2.7 Antioxidant index detection

[0063] 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.

[0064] 2 Results

[0065] 2.1 Identification of the physicochemical properties of ESO-Pm-Bb vaccine emulsion

[0066] 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).

[0067] Table 1 Physicochemical properties of ESO-Pm-Bb vaccine emulsions prepared under different homogenization pressures

[0068]

[0069] 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.

[0070] 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%.

[0071] 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).

[0072] 2.2 Immunization effect of ESO on Pasteurella multocida-Bordetella bronchiseptica inactivated vaccine

[0073] 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

[0074] 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 2As 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).

[0075] 2.2.2 Detection of spleen T lymphocyte subsets and lymphocyte proliferation assay

[0076] 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).

[0077] 2.2.3 Detection of cytokine levels in mouse serum and lymphocyte supernatant

[0078] 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 4In 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).

[0079] 2.2.4 Effects of ESO-Pm-Bb on the protection rate and lung bacterial load in mice after challenge

[0080] 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).

[0081] 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).

[0082] 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).

[0083] 2.2.5 Detection of serum antioxidant indicators before and after virus challenge

[0084] 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 6 (A), 7 days after Pm infection ( Figure 6 7 days after the challenge with B) and Bb FX-1 ( Figure 6In the ESO group, the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) activities in the serum of mice were significantly higher than those in the antigen group and the aluminum adjuvant group. P <0.0001 or P <0.001). Conversely, the content of malondialdehyde (MDA), which represents the degree of lipid peroxidation damage, was significantly reduced in the ESO group ( P <0.05). This indicates that ESO adjuvants can effectively enhance the body's overall antioxidant status and reduce oxidative stress damage that may occur during the immune and infection processes.

[0085] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

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. The volume ratio of the antigen aqueous phase to soybean oil is 1:1, and the content of epigallocatechin gallate is 5 μg / mL. 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.

2. The preparation method according to claim 1, 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 was identified as Bb FX-1.

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

4. 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.

5. Pasteurella multocida nanovaccine prepared by the preparation method according to any one of claims 1-4.

6. The use of the Pasteurella-Botrytis cinerea nanovaccine as described in claim 5 in the preparation of medicaments for the prevention and / or treatment of Pasteurella multocida and / or Botrytis cinerea.

7. A medicine for the prevention and / or treatment of pasteurellosis and / or bortezomibosis, characterized in that, Including the Pasteurella-Botrytis cinerea nanovaccine as described in claim 5.

Images