A composition of Actinobacillus pleuropneumoniae OMVs in porcines, its preparation method and application

Abstract

This invention provides a composition of Actinobacillus contagious pleuropneumoniae (OMVs) for porcine infectious pleuropneumonia, its preparation method, and its application. It belongs to the field of vaccine preparation technology. This invention uses outer membrane vesicles secreted by Actinobacillus contagious pleuropneumoniae type 1 GZ strain and type 7 ZQ strain to synergistically prepare a bivalent inactivated vaccine for porcine infectious pleuropneumonia (OPVC) (type 1 GZ + type 7 ZQ strain). The bivalent vaccine prepared by this invention has a significant preventive effect against porcine infectious pleuropneumonia. Furthermore, the bivalent inactivated vaccine for porcine infectious pleuropneumonia with added outer membrane vesicles, while reducing the inactivated bacterial content of OMVs of type 1 GZ and type 7 ZQ strains, still has a significant preventive effect against porcine infectious pleuropneumonia. It can be used to prepare products for the prevention of porcine infectious pleuropneumonia, thus applying it to the prevention of porcine infectious pleuropneumonia and avoiding bacterial resistance, possessing good practical application value. It also provides a reference for future in-depth research on outer membrane vesicle vaccines.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, and more specifically to a composition of Actinobacillus pleuropneumoniae OMVs in pigs, its preparation method, and its application. Background Technology

[0002] Porcine infectious pleuropneumonia (PPH) is a highly contagious and fatal respiratory disease caused by Actinobacillus pleuropneumoniae (Apn), characterized by acute hemorrhage and chronic fibrinous necrotizing pleuritis. All pig species are susceptible, and outbreaks are often acute in newly introduced herds, with morbidity and mortality rates frequently exceeding 20%, and mortality rates reaching 80%–100% in the most acute form. The disease typically progresses chronically, with affected pigs exhibiting chronic emaciation or developing secondary diseases leading to acute death. Asymptomatic or recovered pigs can remain carriers for extended periods, serving as stable sources of infection. PPH is widely distributed. Since Pattison et al. first reported PPH in 1957, it has been found to be prevalent worldwide. Due to its airborne and contact transmission, the disease is more severe in countries and regions with higher levels of intensive pig farming.

[0003] Outer membrane vesicles (OMVs) are products secreted from the surface of Gram-negative bacteria. They are vesicle-like structures formed on the bacterial surface by budding of the outer membrane under certain mechanisms. These structures include the outer membrane and periplasmic components. Most OMVs are spherical, with a diameter of approximately 20–250 nm. In 1959, De et al. first discovered that the culture medium after filtering Vibrio cholerae could induce an immune response against Vibrio cholerae in rabbits. Chatterje et al., while studying the ultrastructure of Vibrio cholerae in the logarithmic growth phase, found vesicles formed by budding of Gram-negative bacterial cell walls in the sterile filtrate of liquid culture medium. Moreover, near sites on the outer membrane where vesicle budding is prone to occur, the content of particulate matter was high. These particles can enter the budding vesicles along with the cytoplasm. The production of OMVs represents a unique mechanism by which bacteria release large amounts of complex protein components and phospholipids into the external environment. Subsequent research revealed that almost all Gram-negative bacteria can produce OMVs. Besides outer membrane proteins, their chemical structure comprises other membrane structural components, hence the term "outer membrane vesicles." The outer membrane components in OMVs can stimulate the body to produce adaptive immune memory, and the LPS they contain can act as an adjuvant. Furthermore, they possess a certain degree of safety as non-replicating vaccines. These factors have made OMVs a popular choice for developing highly effective non-replicating vaccines.

[0004] Currently, outer membrane vesicles are mostly extracted using ultracentrifugation with two ultracentrifugations. However, ultracentrifugation has a limited sample processing capacity, is difficult to operate, costly, time-consuming, and labor-intensive, and cannot extract outer membrane vesicles in large quantities, making it unsuitable for industrial production. This invention overcomes these shortcomings by using a hollow fiber concentration system to concentrate the sample, enabling the extraction of outer membrane vesicles in large quantities. At the same time, PEG concentration is used to replace two ultracentrifugations, which reduces costs and operational difficulty, making the large-scale extraction and industrialization of outer membrane vesicles possible.

[0005] Currently, most vaccines for porcine infectious pleuropneumonia (IPP) are inactivated bacterial vaccines, but the preparation process is cumbersome, costly, and has poor immunization efficacy. OMVs (Organic Vesicle Molecules) possess the ability to mediate the transfer of DNA fragments, autolysins, cytotoxins, virulence factors, and various biomolecules. Their composition makes them important factors in activating the host's innate and adaptive immune response pathways. In addition to the potent immunomodulatory molecule LPS, vesicles also contain OM porin and other important innate immune activating ligands. The immunogenicity of OMVs can lead to protective mucosal and systemic bactericidal antibody responses and has been used in vaccine development. All current experimental evidence indicates that the innate immune response to OMVs is the result of the combined action of bacterial-associated molecular patterns (PAMPs) and LPS recognition. Lipoproteins and OM proteins in vesicles are bioactive molecules that can activate immune cells and induce leukocyte migration.

[0006] Therefore, how to apply outer membrane vesicles to porcine infectious pleuropneumonia vaccines, and provide a vaccine that still has a significant preventive effect against porcine infectious pleuropneumonia while reducing the content of inactivated bacteria, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a composition of Actinobacillus pleuropneumoniae OMVs in pigs, its preparation method and application.

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

[0009] A composition of Actinobacillus pleuropneumoniae OMVs, the composition comprising OMVs secreted by Actinobacillus pleuropneumoniae strain GZ type 1 and OMVs secreted by Actinobacillus pleuropneumoniae strain ZQ type 7, wherein the OMVs are vesicle-like bodies with a double membrane structure, carrying bacterial outer membrane and periplasmic components.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned porcine infectious pleuropneumoniae Actinobacillus OMVs composition, comprising the following steps:

[0011] (1) The type 1 GZ strain and the type 7 ZQ strain of Actinobacillus pleuropneumoniae were fermented and cultured for 8-10 hours respectively to prepare the fermentation broth of type 1 GZ strain and the fermentation broth of type 7 ZQ strain.

[0012] (2) Centrifuge the two fermentation liquids mentioned above, take the supernatant, concentrate it through a hollow fiber system, and prepare the supernatant concentrate of type 1 GZ strain and the supernatant concentrate of type 7 ZQ strain.

[0013] (3) Take the above two supernatant concentrates and add PEG to concentrate at a rate of 6-14%, let stand, centrifuge and separate, take the precipitates and add PBS to resuspend and filter to prepare type 1 GZ strain OMVs and type 7 ZQ strain OMVs, mix them to prepare a composition of porcine infectious pleuropneumoniae Actinobacillus OMVs.

[0014] Preferably, the fermentation conditions in step (1) are as follows: fermentation temperature 37℃, stirring speed 100-200rpm, pH 7.2-7.4, fermentation tank pressure 0.03-0.05pa, and fermentation time 8-10h.

[0015] Preferably, the culture medium for fermentation in step (1) is a TSB medium containing NAD and newborn calf serum, wherein the final concentration of NAD in the culture medium is 0.01% and the final concentration of newborn calf serum is 2%.

[0016] Preferably, the centrifugation process in step (2) is as follows: 2-8℃, 8000rpm, centrifugation for 15min; the concentration is carried out using a 300KDa hollow fiber system.

[0017] Preferably, in step (3), the amount of PEG added is 6-14% of the total weight of the supernatant concentrate, and the mixture is stirred at 2-8℃ for 4 hours. The settling time is 18-24 hours. The centrifugation process is as follows: 12000 rpm, 4℃, centrifugation for 30 minutes. The filtration is carried out using a 0.22μm filter membrane.

[0018] Another object of the present invention is to provide the use of the above-described OMVs composition or the OMVs composition prepared by the above method in the preparation of porcine infectious pleuropneumonia vaccine or vaccine adjuvant.

[0019] Another object of the present invention is to provide a bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia, said bivalent inactivated OMV vaccine comprising the above-mentioned porcine infectious pleuropneumonia Actinobacillus OMVs composition.

[0020] Preferably, the bivalent inactivated OMV vaccine further includes inactivated strains of Actinobacillus pleuropneumoniae type 1 GZ strain and type 7 ZQ strain.

[0021] Another object of the present invention is to provide a method for preparing a bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia, comprising weighing the raw materials according to the following weight proportions and preparing the vaccine:

[0022] (1) Preparation of oil phase: Take 94 parts of white oil for injection, add 6 parts of Span-80, and then add 2 parts of aluminum stearate while stirring. Heat and stir until transparent, sterilize at high temperature and high pressure to prepare the oil phase for later use.

[0023] (2) Preparation of aqueous phase: Based on the results of viable count and BCA content detection of outer membrane vesicles, and according to requirements, the inactivated bacterial solution of Actinobacillus pleuropneumoniae type 1 GZ strain, the inactivated bacterial solution of type 7 ZQ strain, and the composition of Actinobacillus pleuropneumoniae OMVs in porcine infectious pleuropneumoniae were mixed in different proportions to prepare a mixed bacterial solution containing outer membrane vesicles. Take 96 parts of the mixed bacterial solution, sterilize 4 parts with Tween-80, mix thoroughly, and prepare an aqueous phase for later use.

[0024] (3) Emulsification: Take 2 parts of the oil phase and stir at low speed while slowly adding 1 part of the water phase. Stir at 4000 rpm for 30 minutes to emulsify into an oil emulsion. Before stopping the stirring, add 1% thimerosal solution so that its final concentration in the vaccine does not exceed 0.01%, thus obtaining the bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia.

[0025] Preferably, the concentrations of the inactivated Actinobacillus pleuropneumoniae type 1 GZ strain bacterial solution and the inactivated Actinobacillus pleuropneumoniae type 7 ZQ strain bacterial solution in step (2) are both ≥3.2×10⁻⁶. 9 CFU / mL, the two bacterial solutions were mixed in equal volumes, and the content of both OMVs in the composition was ≥78.125μg / mL.

[0026] Preferably, the concentration of the outer membrane vesicles of Actinobacillus pleuropneumoniae type 1 GZ strain and type 7 ZQ strain in the bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia is 25 μg / ml.

[0027] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) Animal experiments have demonstrated that the bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia of the present invention has a significant effect in preventing porcine infectious pleuropneumonia.

[0029] (2) The bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia prepared by the vaccine still has a significant effect in preventing porcine infectious pleuropneumonia even when the content of inactivated bacteria of Actinobacillus pleuropneumoniae type 1 GZ strain and type 7 ZQ strain is reduced.

[0030] (3) The present invention uses a hollow fiber concentration system to concentrate the sample, which can realize the large-scale extraction of outer membrane vesicles. At the same time, PEG concentration is used to replace two ultracentrifugations, which reduces the cost and the difficulty of operation, making the large-scale extraction and industrialization of outer membrane vesicles possible. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0032] Figure 1 The attached image shows the preparation process of Actinobacillus pleuropneumoniae OMVs.

[0033] Figure 2 The attached images are transmission electron microscope (TEM) images of outer membrane vesicles of strain GZ and strain ZQ.

[0034] Figure 3 The attached figure is a standard curve of protein concentration versus absorbance.

[0035] Figure 4 The attached image shows the electrophoresis results of OMVs proteins.

[0036] Figure 5 The attached figure shows the particle size detection results of outer membrane vesicles in strain GZ.

[0037] Figure 6 The attached figure shows the particle size detection results of the outer membrane vesicles of strain ZQ. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1

[0040] Activation and propagation of strains

[0041] (1) Strain selection and activation: freeze-dried serological strain GZ of Actinobacillus pleuropneumoniae type 1 (deposited at China General Microbiological Culture Collection Center, accession number CGMCC NO.45994, accession date June 24, 2024, taxonomic name: Actinobacillus pleuropneumoniae type 1) and strain ZQ of type 7 (deposited at China General Microbiological Culture Collection Center, accession number CGMCC NO.46086, accession date June 24, 2024, taxonomic name: Actinobacillus pleuropneumoniae type 7) were obtained.

[0042] After dissolving in tryptone soybean broth (TSB), the culture was inoculated onto TSA slant agar containing 0.01% NAD and 2% newborn calf serum, and incubated at 37°C with a CO2 concentration of 5%–10% for 24 hours. The culture was then streaked onto TSA agar plates containing 0.01% NAD and 2% newborn calf serum and incubated at 37°C for 16–18 hours. Then, 5–10 typical colonies were selected from each culture and inoculated onto TSA slant agar plates containing 0.01% NAD and 2% newborn calf serum, and incubated at 37°C with a CO2 concentration of 5%–10% for 16–18 hours. After passing the purity test, the culture was used as the primary seed culture.

[0043] (2) Expanding culture of the strain: The primary seed was inoculated into tryptone soybean broth (TSB) medium containing 0.01% NAD and 2% newborn calf serum, and cultured at 37°C with shaking for 8-10 hours. After passing the purity test, the sample was used as the secondary seed.

[0044] Example 2

[0045] Preparation of a composition of Actinobacillus pleuropneumoniae OMVs in porcine organisms

[0046] (1) Take the secondary seed liquid of the strain prepared in Example 1 and culture it separately in a bio-fermenter. Add TSB medium at 60% to 70% of the total volume of the bio-fermenter. At the same time, add 0.02% of defoamer according to the amount of medium to defoam. When the temperature of TSB medium drops to about 37°C, add NAD and newborn calf serum at 0.01% and 2% of the total amount of medium, respectively. At the same time, inoculate the secondary seed liquid of strain GZ and strain ZQ at 3% to 5% of the total amount of medium. Set the temperature to 37°C, the stirring speed to 100 to 200 rpm, the pH value to 7.2 to 7.4, the infusion pressure to 0.03 to 0.05 Pa, and the culture time to 8 to 10 hours.

[0047] (2) After separate fermentation culture, the fermentation broths of porcine infectious pleuropneumonia GZ strain and ZQ strain were obtained. The broths were centrifuged at 8000 rpm and 4℃ for 15 min. The supernatant was collected and the precipitate was discarded. The supernatant was concentrated through a 300 kDa hollow fiber system to retain OMVs in the concentrate, and finally the supernatant concentrate was obtained.

[0048] (3) The supernatant concentrates of the two strains were added to PEG, stirred at 2-8℃ for 4 hours, and then allowed to stand for 18-24 hours. After centrifugation at 12000 rpm and 4℃ for 30 minutes, the supernatant was discarded, the precipitate was resuspended with PBS buffer, and filtered through a 0.22 μm filter membrane to obtain the outer membrane vesicles of porcine infectious pleuropneumonia strains GZ and ZQ, respectively. These were then mixed to prepare the Actinobacillus pleuropneumoniae OMVs composition. OMVs are vesicle-like bodies with a double membrane structure, carrying bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens, and various pathogen-related molecular patterns. They were stored at -80℃ for later use. The preparation process is as follows: Figure 1 As shown.

[0049] Example 3

[0050] Analysis of OMVs of Actinobacillus contagious pleuropneumoniae in porcines

[0051] (1) TEM characterization: The uniformly dispersed outer membrane vesicles of strain GZ and strain ZQ prepared in Example 2 were diluted to 500-800 μg / ml, respectively. 10 μL of the diluted solution was dropped onto a copper grid, and after adsorption for half an hour, it was blotted dry with filter paper. Subsequently, 10 μL of 1% phosphotungstic acid was dropped onto each vesicle for 3-5 min, and then blotted dry with filter paper again. The size and morphology of OMVs derived from strains GZ and ZQ of Actinobacillus pleuropneumoniae were observed by TEM. TEM revealed complete shapes and regular spherical structures with particle sizes within 200 nm. The transmission electron microscopy images of the OMVs are shown below. Figure 2 As shown.

[0052] (2) OMVs protein quantification: OMVs protein concentration was determined using the BCA method. Following the standard procedures in the instruction manual, a protein concentration-absorbance standard curve was established using protein standards (e.g., ...). Figure 3 ), R 2 =0.9944>0.99, showing a good linear relationship; 25 μL of OMVs sample diluted 10 times was added to two sub-wells, followed by 200 μL of BCA working solution, and incubated at 37℃ for 30 min. The absorbance was then measured at a wavelength of 562 nm.

[0053] Results analysis: Calculations showed that the OMVs protein concentrations of strain GZ and strain ZQ were 10.452 mg / mL and 16.599 mg / mL, respectively, indicating high yields suitable for industrial production.

[0054] (3) OMV protein types: Take 80 μL each of GZ strain and ZQ strain OMV solutions diluted to 500-800 μg / ml, mix with 20 μL of 5× protein loading buffer, and boil for 10 min. For each type, take 10 μL, 20 μL of the boiled mixed sample, and 5 μL of Mark, respectively, and load them onto 12% SDS-PAGE. Electrophoresis is performed at 80V for 20 min, followed by electrophoresis at 100V for 30 min. Then, Coomassie brilliant blue staining is performed, followed by destaining with destaining solution. The electrophoresis results are as follows. Figure 4 As shown.

[0055] Results analysis: such as Figure 4 The SDS-PAGE electrophoresis of Actinobacillus pleuropneumoniae strain GZ and ZQ OMVs showed multiple protein bands, with the size of the protein bands mainly between 25 kDa and 200 kDa.

[0056] (4) Particle size detection: After the nanoparticle size and zeta potential meter was preheated for 30 minutes, 1 mL of sample (at a concentration of not less than 25 μg / mL) was added to each sample cup. The sample cup was placed in the sample chamber, and the "size" mode was selected to enter the particle size measurement interface for detection. The results are as follows: Figure 5 (Type 1 GZ strain) and Figure 6 (Type 7 ZQ strain) is shown.

[0057] Results analysis: The average particle size of strain GZ was 82.62 nm, and the average particle size of strain ZQ was 78.38 nm.

[0058] Example 4

[0059] Preparation of a bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia according to the present invention

[0060] (1) Oil phase preparation

[0061] Take 94 parts of white oil for injection, add 6 parts of Span-80, then add 2 parts of aluminum stearate while stirring, heat and stir until transparent, sterilize at high temperature and high pressure to prepare the oil phase for later use.

[0062] (2) Aqueous phase preparation

[0063] a: Preparation of Actinobacillus pleuropneumoniae bacterial suspension:

[0064] Actinobacillus pleuropneumoniae strain 1 GZ and strain 7 ZQ were cultured separately in fermenters until they reached the stationary phase, and bacterial cultures were obtained separately.

[0065] After the bacterial culture was completed, samples were taken and tested for purity using tryptone soy agar (TSA) medium containing 0.01% NAD and 2% serum. Simultaneously, viable cell counts were performed on samples, with each culture containing ≥4.5 × 10⁻⁶ viable cells.9 CFU / ml.

[0066] Slowly add formaldehyde solution at 0.3% of the total bacterial volume, mix thoroughly, inactivate at 37°C for 24 hours, stirring 4-6 times during the process. After passing the inactivation test, store the bacterial solution at 2-8°C for later use.

[0067] b: Preparation of aqueous phase by mixing two bacterial cultures and a composition of Actinobacillus pleuropneumoniae OMVs:

[0068] Based on the results of viable cell count and BCA content detection of outer membrane vesicles, the two bacterial solutions and the Actinobacillus pleuropneumoniae OMVs composition were mixed (Note: OMV + 1 / 2 whole cell group vaccine was prepared by mixing the two bacterial solutions to ≥1.6×10). 9 After CFU / ml was collected, equal volumes were mixed, and the content of the two OMVs in the composition was ≥78.125 μg / ml; for the OMV+1 / 3 whole cell group vaccine, the two bacterial solutions were prepared to have a concentration ≥1.09×10⁻⁶. 9 After mixing equal amounts of CFU / ml, and ensuring the content of the two OMVs in the composition is ≥78.125μg / ml, a mixed bacterial solution of outer membrane vesicles is prepared. Take 96 portions of the mixed bacterial solution of outer membrane vesicles, sterilize 4 portions at Tween-80, mix thoroughly, and prepare an aqueous phase for later use.

[0069] (3) Emulsification

[0070] While stirring at low speed, slowly add 1 part of the aqueous phase and stir at 4000 rpm for 30 minutes to emulsify into an oil emulsion. Before stopping stirring, add 1% thimerosal solution to ensure that the final concentration does not exceed 0.01%, thus obtaining the bivalent inactivated OMV vaccine for porcine infectious pleuropneumonia.

[0071] The sterility test shall be conducted in accordance with the appendix of the current Chinese Veterinary Pharmacopoeia, and no sterile growth should be observed.

[0072] Example 5

[0073] Preparation of bivalent inactivated vaccine for porcine contagious pleuropneumonia (type 1 GZ strain + type 7 ZQ strain), i.e., whole cell vaccine

[0074] (1) Oil phase preparation

[0075] Take 94 parts of white oil for injection, add 6 parts of Span-80, then add 2 parts of aluminum stearate while stirring, heat and stir until transparent, sterilize at high temperature and high pressure to prepare the oil phase for later use.

[0076] (2) Aqueous phase preparation

[0077] a: Preparation of Actinobacillus pleuropneumoniae bacterial suspension:

[0078] Actinobacillus pleuropneumoniae strain 1 GZ and strain 7 ZQ were cultured separately in fermenters until they reached the stationary phase, and bacterial cultures were obtained separately.

[0079] After the bacterial culture was completed, samples were taken and tested for purity using tryptone soy agar (TSA) medium containing 0.01% NAD and 2% serum. Simultaneously, viable cell counts were performed on samples, with each culture containing ≥4.5 × 10⁻⁶ viable cells. 9 CFU / ml.

[0080] Slowly add formaldehyde solution at 0.3% of the total bacterial volume, mix thoroughly, inactivate at 37°C for 24 hours, stirring 4-6 times during the process. After passing the inactivation test, store the bacterial solution at 2-8°C for later use.

[0081] b: Preparation of aqueous phase mixture of two types of Actinobacillus pleuropneumoniae:

[0082] Based on the viable cell count results, the two bacterial solutions (prepared to a concentration of no less than 3.2 × 10⁻⁶) were tested. 9 (After mixing equal amounts of CFU / ml), take 96 portions of the mixed bacterial solution, sterilize 4 portions at Tween-80, and mix thoroughly. Prepare the aqueous phase for later use.

[0083] (3) Emulsification

[0084] While stirring at low speed, slowly add 1 part of the aqueous phase and stir at 4000 rpm for 30 minutes to emulsify into an oil emulsion. Before stopping stirring, add 1% thimerosal solution to ensure that the final concentration does not exceed 0.01%, thus obtaining the bivalent inactivated vaccine for porcine infectious pleuropneumonia.

[0085] The sterility test shall be conducted in accordance with the appendix of the current Chinese Veterinary Pharmacopoeia, and no sterile growth should be observed.

[0086] Example 6

[0087] Swine infectious pleuropneumonia bivalent inactivated OMV vaccine immunization challenge test

[0088] (1) Grouping of immune challenge experiment

[0089] The seedlings used in the experiment were prepared according to the contents listed in Table 1 and the method described in Example 4.

[0090] (2) Immunity

[0091] Sixty healthy, susceptible piglets aged 5–6 weeks (APP indirect hemagglutination titer ≤ 1:4) were selected and divided into 14 groups (as listed in Table 1), with 5 piglets in each group. Immunization was performed according to the groups in Table 1, with groups 13–14 serving as controls. Each piglet was injected with the same dose of sterile physiological saline. Note: The whole-cell vaccine was prepared according to the method described in Example 5.

[0092] (3) Attacking the poison

[0093] Twenty-eight days after immunization, each immunization group, along with the control group, was injected intratracheally with 2.0 ml of the minimum symptomatic dose (virus testing required before challenge) of either the GZ or ZQ strain of highly virulent bacteria, according to the experimental grouping (see Table 1, Immune Challenge Grouping Table). The results were observed for 14 days and are shown in Table 2.

[0094]

[0095]

[0096]

[0097]

[0098] Results Analysis: As can be seen from the results of the virus challenge in Table 2:

[0099] 1. OMV vaccines with added bacteria offer higher protection than OMV vaccines alone;

[0100] 2. The vaccines with the addition of two types of OMV bacteria showed significantly higher protection rates compared to those without.

[0101] 3. The vaccine with two types of OMV bacteria added has a significantly higher protection rate than the vaccines with only 1 / 2 bacteria and 1 / 3 bacteria bacteria.

[0102] In summary, the bivalent inactivated swine contagious pleuropneumonia vaccine (GZ strain 1 + ZQ strain 7) containing OMV of swine contagious pleuropneumonia type 1 (GZ strain) and type 7 (ZQ strain) provides protection for animals and significantly enhances their immune efficacy. Furthermore, it maintains excellent immunization even with reduced bacterial count, thus lowering costs while improving vaccine quality. Therefore, it is indeed a viable candidate for development and application as a novel vaccine.

[0103] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A porcine infectious pleuropneumoniae Actinobacillus ( Actinobacillus pleuropneumoniae The bivalent combined vaccine is characterized by, It is prepared by emulsifying an aqueous phase and an oil phase, wherein the aqueous phase comprises: (a) Inactivated bacterial cells of Actinobacillus pleuropneumoniae type 1 GZ strain in pigs; (b) Inactivated bacterial cells of Actinobacillus pleuropneumoniae strain 7 ZQ from pigs; (c) A composition of Actinobacillus pleuropneumoniae OMVs consisting of OMVs secreted by Actinobacillus pleuropneumoniae type 1 GZ strain and OMVs secreted by Actinobacillus pleuropneumoniae type 7 ZQ strain; The preservation number of the type 1 GZ strain is CGMCC NO.45994, and the preservation number of the type 7 ZQ strain is CGMCC NO.46086.

2. The bivalent combined vaccine against Actinobacillus pleuropneumoniae in porcines according to claim 1, characterized in that, The porcine infectious pleuropneumoniae Actinobacillus OMVs composition was prepared using the following method: (1) The type 1 GZ strain and the type 7 ZQ strain of Actinobacillus pleuropneumoniae were fermented and cultured to prepare fermentation broth of type 1 GZ strain and type 7 ZQ strain, respectively. (2) The two fermentation liquids were centrifuged and separated, and the supernatant was taken and concentrated through a hollow fiber system to prepare the supernatant concentrate of type 1 GZ strain and the supernatant concentrate of type 7 ZQ strain. (3) Take the above two supernatant concentrates and add PEG to concentrate at a rate of 6-14%, let stand, centrifuge and separate, take the precipitates and add PBS to resuspend and filter to prepare type 1 GZ strain OMVs and type 7 ZQ strain OMVs, mix them to prepare a composition of porcine infectious pleuropneumonia actinobacillus OMVs.

3. The bivalent combined vaccine against Actinobacillus pleuropneumoniae of porcine contagious pleuropneumoniae according to claim 2, characterized in that, The fermentation conditions in step (1) are as follows: fermentation temperature 37℃, stirring speed 100-200rpm, pH 7.2-7.4, fermentation tank pressure 0.03-0.05pa, and fermentation time 8-10h; the fermentation medium in step (1) is TSB medium containing NAD and newborn calf serum, wherein the final concentration of NAD in the medium is 0.01% and the final concentration of newborn calf serum is 2%.

4. The bivalent combined vaccine against Actinobacillus pleuropneumoniae of porcine contagious pleuropneumoniae according to claim 2, characterized in that, The centrifugation process in step (2) is as follows: centrifuge at 2-8℃, 8000rpm for 15min; the concentration is carried out using a 300KDa hollow fiber system; the amount of PEG added in step (3) is 6-14% of the total weight of the supernatant concentrate, and the mixture is stirred at 2-8℃ for 4h; the settling time is 18-24h; the centrifugation process is as follows: centrifuge at 12000rpm, 4℃ for 30min; the filtration is carried out using a 0.22μm filter membrane.

5. The bivalent combined vaccine against Actinobacillus pleuropneumoniae of porcine contagious pleuropneumoniae according to claim 1, characterized in that, The concentration of outer membrane vesicles of both Actinobacillus pleuropneumoniae type 1 GZ strain and type 7 ZQ strain in the bivalent combined vaccine for porcine infectious pleuropneumoniae was 25 μg / ml.

6. A method for preparing a bivalent combined vaccine against Actinobacillus pleuropneumoniae of porcine contagious pleuropneumoniae according to any one of claims 1-5, characterized in that, Weigh the raw materials according to the following proportions and prepare them accordingly: (1) Preparation of oil phase: Take 94 parts of white oil for injection, add 6 parts of Span-80, and then add 2 parts of aluminum stearate while stirring. Heat and stir until transparent, sterilize at high temperature and high pressure to prepare the oil phase for later use. (2) Preparation of aqueous phase: Based on the results of viable bacterial count and BCA content detection of outer membrane vesicles, and according to requirements, the inactivated bacterial solution of Actinobacillus pleuropneumoniae type 1 GZ strain, the inactivated bacterial solution of type 7 ZQ strain, and the composition of Actinobacillus pleuropneumoniae OMVs in porcine infectious pleuropneumoniae were mixed in different proportions to prepare a mixed bacterial solution containing outer membrane vesicles. 96 parts of the mixed bacterial solution were sterilized with Tween-80 for 4 parts, and the mixture was thoroughly mixed to prepare an aqueous phase for later use. (3) Emulsification: Take 2 parts of the oil phase and stir at low speed while slowly adding 1 part of the water phase. Stir at 4000 rpm for 30 minutes to emulsify into an oil emulsion. Before stopping the stirring, add 1% thimerosal solution so that its final concentration in the vaccine does not exceed 0.01%, thus obtaining the bivalent combined vaccine of Actinobacillus pleuropneumoniae in pigs.

7. The preparation method according to claim 6, characterized in that, The concentrations of the inactivated strains of Actinobacillus pleuropneumoniae type 1 GZ and type 7 ZQ described in step (2) are both ≥3.2×10⁻⁶. 9 CFU / mL, the two bacterial solutions were mixed in equal volumes, and the content of both OMVs in the composition was ≥78.125μg / mL.

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