CN118879559A - Outer membrane vesicles of Haemophilus parasuis type 12 strain JS1015 and preparation method and application thereof - Google Patents

Abstract

The invention discloses a haemophilus parasuis outer membrane vesicle, and relates to the technical field of vaccine preparation, wherein the outer membrane vesicle is secreted by a haemophilus parasuis serotype 12 JS1015 strain. According to the invention, the haemophilus parasuis outer membrane vesicles are used as vaccines for immunizing animals for the first time, animal experiments prove that the haemophilus parasuis outer membrane vesicles have an obvious effect of preventing haemophilus parasuis diseases, and can be used for preparing products for preventing haemophilus parasuis diseases, so that the haemophilus parasuis outer membrane vesicles are applied to preventing haemophilus parasuis diseases, and avoid bacterial drug resistance, and have good practical application values.

Description

An outer membrane vesicle of Haemophilus parasuis type 12 JS1015 strain and its preparation method and application

Technical Field

The invention relates to the technical field of Haemophilus parasuis outer membrane vesicle preparation and vaccine preparation, and more specifically to Haemophilus parasuis outer membrane vesicle and a preparation method and application thereof.

Background Art

At present, the understanding of the pathogenesis of Haemophilus parasuis infection is relatively limited, and preventive measures still cannot completely control the occurrence of the disease, making Haemophilus parasuis disease a major problem in the pig farming industry. Haemophilus parasuis disease is usually controlled by antimicrobial treatment and vaccination. However, it has been reported that clinical isolates of Haemophilus parasuis are resistant to antibiotics, so vaccination and immune antibodies (IgG or IgA) are generally considered to be effective means of controlling Haemophilus parasuis disease. However, due to the large number of serotypes of Haemophilus parasuis and the fact that a large proportion of strains have not been typed, it is extremely difficult to develop an efficient cross-protective vaccine. The effects and research of many vaccines still have limitations. The types of Haemophilus parasuis vaccines that have been reported include inactivated vaccines, subunit vaccines, DNA vaccines, and attenuated vaccines. At present, the only commercial vaccines used in the prevention and control of Haemophilus parasuis disease worldwide are inactivated vaccines and subunit vaccines. Although vaccination can reduce mortality, vaccine failure is also common due to its low protective effect or poor cross-protection. Outer membrane vesicles (OMVs) have the properties of mediating the transfer of DNA fragments, autolytic enzymes, cytotoxins, virulence factors, and a variety of biomolecules. The composition of OMVs makes them an important factor in activating the host's innate and acquired immune response pathways. In addition to the powerful immunomodulatory molecule LPS, the vesicles also contain O M porins 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 shows that the innate immune response to OMVs is the result of the combined effects of bacteria-associated molecular patterns (PAMPs) and the recognition of LPS. The lipoproteins and OM proteins in the vesicles are bioactive molecules that can activate immune cells and induce leukocyte migration.

In recent years, the systemic inflammatory diseases caused by Haemophilus parasuis have caused huge economic losses to the global pig industry. How to prevent and control the occurrence of Haemophilus parasuis has become a focus of attention, and the research and development of vaccines has also received attention.

Outer membrane vesicles (OMVs) are products secreted from the surface of Gram-negative bacteria. They are formed by the outer membrane of bacteria budding under certain mechanisms and forming a vesicle-like structure on the surface of bacteria. This structure includes the outer membrane and periplasmic components. Most outer membrane vesicles are spherical, with a diameter of about 20 to 250 nm. In 1959, De et al. first discovered that the culture medium of Vibrio cholerae after filtering out bacteria could induce an immune response to Vibrio cholerae in rabbits. Chatterje et al. studied the ultrastructure of Vibrio cholerae in the logarithmic growth period and found that there were vesicles formed by budding of the cell wall of Gram-negative bacteria in the sterile filtrate of the liquid culture medium. In addition, the content of particulate matter was high near the site where the outer membrane was prone to vesicle budding. These particles can enter the budding vesicles with the cytoplasm. The production of OMVs represents that bacteria have a unique mechanism to release a large amount of complex protein components and phospholipids to the external environment. Later studies found that almost all Gram-negative bacteria can produce OMVs. Their chemical structure is composed of outer membrane proteins and other membrane structural components, so people call this type of membrane structure vesicles outer membrane vesicles.

With the progress of public health, the incidence of bacterial infections has decreased, but bacterial infections are still one of the main causes of human illness and death. Due to the increase and rapid spread of drug-resistant bacteria, the effectiveness of antibiotics in treating bacterial diseases has been challenged. Therefore, vaccines are considered to be the most direct and effective strategy to deal with bacterial diseases in the post-antibiotic era. An efficient vaccine must have three major factors: (1) antigen: able to trigger an immune response and produce immune memory to prevent the body from further attack; (2) adjuvant: to ensure that the immune response is strong enough; (3) have a certain degree of safety. The outer membrane components contained in OMVs can stimulate the body to produce adaptive immune memory, the LPS contained can serve as a self-adjuvant, and as a non-replicating vaccine, it has a certain degree of safety. Therefore, these factors make OMVs a popular choice for the development of non-replicating and efficient vaccines.

At present, outer membrane vesicles are mostly extracted by ultracentrifuging twice in an ultracentrifuge. However, the ultracentrifuge can only process a limited amount of samples, is difficult to operate, is costly, time-consuming and labor-intensive, and cannot extract a large amount of outer membrane vesicles, making it unsuitable for industrial production. The present invention overcomes this defect. The present invention uses a hollow fiber concentration system to concentrate the sample, which can achieve a large amount of outer membrane vesicles extraction. At the same time, PEG concentration is used instead of two ultracentrifugations, which reduces both the cost and the difficulty of operation, making the large-scale extraction and industrialization of exosomes possible.

Therefore, how to provide an outer membrane vesicle of Haemophilus parasuis with good immunogenicity and simple preparation process and prepare it into a vaccine is a technical problem that technical personnel in this field urgently need to solve.

Summary of the invention

In view of this, the present invention provides an outer membrane vesicle secreted by Haemophilus parasuis type 12 JS1015 strain, and records its preparation method and application. The output of the vaccine of the present invention enhances the immune effect, produces good cross-immunity, improves the protection rate of the vaccine to animals, avoids the current situation that bacterial diseases caused by bacterial resistance are difficult to control, and realizes effective prevention of Haemophilus parasuis disease.

In order to achieve the above object, the present invention adopts the following technical solution:

An outer membrane vesicle of Haemophilus parasuis, wherein the outer membrane vesicle is secreted by Haemophilus parasuis serotype 12 type JS1015 strain; the outer membrane vesicle is composed of bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens and multiple pathogen-related molecular patterns. The Haemophilus parasuis serotype 12 type JS1015 strain has a preservation number of CGMCC No. 14418 and is classified as Haemophilus parasuis.

In the above technical solution, the outer membrane vesicle particle size is within 200nm, the protein band size is concentrated between 25KDa-200 KDa, and each outer membrane vesicle is composed of bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial specific antigens and a variety of pathogen-related molecular patterns. The outer membrane component can stimulate the body to produce adaptive immune memory, the LPS contained can be used as a self-adjuvant, and as a non-replicating vaccine, it has a certain safety. The vesicle also contains OM pore protein and other important innate immune activation ligands.

Preferably, the composition of OMVs makes them important factors in activating the host's innate and adaptive immune response pathways. In addition to the powerful immunomodulatory molecule LPS, the vesicles also contain O M porins and other important innate immune activating ligands.

As the same inventive concept as the above technical solution, the present invention also claims protection for a method for preparing the above outer membrane vesicles, comprising the following steps:

(1) fermenting Haemophilus parasuis serotype 12 strain JS1015 to a stable phase to obtain a bacterial solution;

(2) Centrifuge the bacterial solution at 10,000 rpm/min, 2-8°C for 10 min, and concentrate the supernatant through a 300 kd hollow fiber system to obtain a concentrate;

(3) 7% PEG was added to the concentrate, stirred at 2-8°C for 4 h, allowed to stand at 2-8°C overnight, and then centrifuged at 12000 rpm/min for 30 min. The precipitate was resuspended in PBS and filtered through a 0.22 μm filter to obtain the outer membrane vesicles of Haemophilus parasuis.

In the above technical scheme, in step (1), the fermentation culture is to control the dissolved oxygen to 33-36% by adjusting the air intake and the stirring speed; the fermentation culture is carried out at 37°C for 10-12 hours; the culture medium for the fermentation culture is TSB, to which NAD with a final concentration of 4 μg/ml and 5% newborn calf serum are added.

The present invention also claims to protect the use of the outer membrane vesicles in preparing a vaccine for preventing Haemophilus parasuis disease.

The present invention also claims a vaccine for preventing Haemophilus parasuis disease, comprising the outer membrane vesicles of Haemophilus parasuis.

In the vaccine, the concentration of the outer membrane vesicles of Haemophilus parasuis is 0.05-20 mg/mL.

The present invention uses the outer membrane vesicles of Haemophilus parasuis as a vaccine for immunizing animals for the first time. Animal experiments have proved that the outer membrane vesicles of Haemophilus parasuis of the present invention have a significant effect in preventing Haemophilus parasuis disease, can be used to prepare products for preventing Haemophilus parasuis disease, and thus are applied to preventing Haemophilus parasuis disease and avoiding bacterial resistance, and have good practical application value.

The present invention uses a hollow fiber concentration system for concentration, which can achieve large-scale extraction of outer membrane vesicles. At the same time, PEG concentration is used instead of two ultracentrifugations, which not only reduces the cost but also reduces the difficulty of operation, making large-scale extraction and industrialization of exosomes possible.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

Figure 1 is a flow chart of the preparation of outer membrane vesicles OMVs;

Figure 2 is a transmission electron micrograph of the OMVs of Haemophilus parasuis type 12 JS strain in Example 3;

Figure 3 is a standard curve of protein concentration-absorbance of Haemophilus parasuis type 12 JS strain OMVs in Example 3;

Figure 4 is a protein SD S-PAGE electrophoresis diagram of the OMVs of Haemophilus parasuis type 12 JS strain in Example 3;

FIG5 is a graph showing the particle size detection results of OMVs of Haemophilus parasuis type 12 JS strain in Example 3.

DETAILED DESCRIPTION

The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values should be understood as modified by the word "approximately" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

Unless otherwise defined, all the professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

The drugs required for the present invention are conventional experimental drugs, which are purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods and will not be described in detail here.

The JS1015 strain of Haemophilus parasuis serotype 12 used in the embodiment is deposited with CGMCC No. 14418 and is classified as Haemophilus parasuis. It was deposited in the General Microbiology Center of China Culture Collection Administration on July 13, 2017, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The serotype 4 strain of Haemophilus parasuis SH1012 is deposited with the CGMCC No.14417 and is classified as Haemophilus parasuis. It was deposited at the Center for General Microbiology, China Culture Collection on July 13, 2017, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The GD1022 strain type 5 is deposited with the CGMCC No.14419 and is classified as Haemophilus parasuis. It was deposited at the General Microbiology Center of China Culture Collection Administration on July 13, 2017, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Example 1 Selection and cultivation of strains

Strain selection and activation: Select Haemophilus parasuis serotype 12 JS1015 strain to prepare outer membrane vesicles and attack strains. Take out the freeze-dried Haemophilus parasuis JS strain, add 1ml sterile saline to dissolve, streak on TSA plate containing 4μg/ml NAD and 5% newborn calf serum, and culture in a 37℃ incubator for 24-48h. Pick out the single colonies that meet the requirements, purify them for one generation, and inoculate them into 3.0ml TSB medium containing 4μg/ml NAD and 5% newborn calf serum. Shake and culture them at 37℃ for 18-24h to obtain the initial bacterial suspension, which can be used as the first-level seed liquid after passing the purity test.

Expansion culture of strains:

1) Take the first-level seed bacterial suspension and inoculate it into TSB culture medium containing 4 μg/ml NAD and 5% newborn calf serum at a ratio of 2%, and culture it at 37° C. with shaking for 10 to 12 hours. If it passes the purity test, it can be used as the second-level seed liquid.

2) The secondary seed liquid is cultured separately in a fermenter through ventilation. Prepare TSB culture medium at about 70% of the volume of the fermenter, and add defoaming agent at 0.02% of the volume of the culture medium. After high-pressure steam sterilization and cooling, add NAD with a final concentration of 4μg/ml and 5% newborn calf serum, and add secondary seed liquid at 2% of the volume of the culture medium. Control the dissolved oxygen to about 35% by adjusting the air intake and stirring speed. Culture at 37℃ for 10-12h and harvest the culture liquid.

Example 2 Extraction of OMVs

Outer membrane vesicles (OMVs) are double-membrane vesicles that are shed from the cell membrane or secreted by the cell. They carry bacterial outer membrane and periplasmic components, including enzymes, virulence factors, bacterial-specific antigens and a variety of pathogen-associated molecular patterns.

The bacterial solution of Haemophilus parasuis serotype 12 JS1015 strain (the culture solution prepared by the method of Example 1) was obtained by aeration fermentation culture, and centrifuged at 10000rpm/min, 4°C for 10min, the supernatant was taken, and the precipitate was discarded. The supernatant was concentrated by a 300KDa hollow fiber system, and the OMVs were trapped in the concentrate, and finally 200mL of the concentrate was obtained. The concentration was stopped, and PEG was added to the concentrate in an amount of 7%, and after stirring at 2-8°C for 4h, it was placed at 2-8°C and stood overnight. The next day, the supernatant treated with PEG was centrifuged at 12000rpm/min, 4°C for 30min, the supernatant was discarded, the precipitate was resuspended with PBS buffer, and filtered with a 0.22μm filter membrane to obtain the outer membrane vesicles of Haemophilus parasuis serotype 12 JS1015 strain, which were stored at -80°C for standby use (see Figure 1).

Example 3 Characterization of OMVs

TEM characterization: The uniformly dispersed OMVs solution in Example 2 was diluted to 500-800 μg/ml, 10 μL of the diluted solution was dropped on the copper mesh, and after adsorption for half an hour, it was blotted with filter paper; then, 10 μL of 1% phosphotungstic acid was dropped on it for staining for 3-5 minutes, and then it was blotted with filter paper, and the size and morphology of the OMVs were observed by TEM. TEM showed a complete shape and a regular spherical structure, and the particle size was within 200 nm, as shown in the transmission electron micrograph of OMVs derived from Haemophilus parasuis serotype 12 JS1015 strain in Figure 2.

OMVs protein quantification: BCA method was used to determine the protein concentration of OMVs. According to the standard steps in the instruction manual, a protein concentration-absorbance standard curve was established using a protein standard (such as the protein concentration-absorbance standard curve shown in Figure 3), R ² = 0.9944>0.99, showing a good linear relationship; 25 μL of 10-fold diluted OMVs sample was added to two secondary wells, and then 200 μL BCA working solution was added, placed at 37°C for 30 minutes, and the absorbance was measured at a wavelength of 562 nm. The OMVs protein concentration was calculated to be 14.465 mg/mL.

OMVs protein types: 80 μL of OMVs solution diluted to 500-800 μg/ml was mixed with 20 μL of 5× protein loading buffer and boiled for 10 min. 5 μL and 10 μL of the boiled mixed sample and 5 μL of Mark were loaded onto 12% SDS-PAGE, respectively, and electrophoresed at 80 V for 20 min, then at 100 V for 30 min, followed by Coomassie brilliant blue staining and decolorization with decolorizing solution. The electrophoresis results are shown in Figure 4 as the protein SDS-PAGE electrophoretogram of the supernatant OMVs of Haemophilus parasuis serotype 12 JS1015 strain. O MVs contain multiple protein bands, and the size of the protein bands is mainly between 25 KDa and 200 KDa.

Particle size detection: The nanoparticle size and zeta potential instrument was preheated for 30 minutes, 1 mL of OMV sample was added to the sample cup, and the sample concentration was not less than 25 μg/mL. The sample cup was placed in the sample slot, and the "size" mode was selected to enter the particle size measurement interface for detection. The results are shown in Figure 5, and the average particle size is 68.23 nm.

Example 4 Preparation of trivalent inactivated vaccine of Haemophilus parasuis (type 4 SH + type 5 GD strain + type 12 JS strain), i.e., whole cell vaccine (corresponding to the whole cell group of Example 6)

(1) Haemophilus parasuis type 4 SH, type 5 GD and type 12 JS strains are cultured separately in a fermenter with ventilation until the stable phase, and bacterial liquids are obtained respectively;

(2) After the bacterial culture is completed, a purity test should be carried out to count the pure and live bacteria.

(3) The inactivated bacterial solution was centrifuged at 12000r/min continuously, and the flow rate was adjusted so that the bacterial solution was retained in the centrifuge chamber for no less than 1 minute; the supernatant was discarded, and the bacterial sludge was resuspended with sterile saline and washed again. According to the live bacterial count results before inactivation, the bacterial sludge was resuspended with sterile saline and the concentration of the bacterial solution was adjusted so that the content of each bacterium was 9.0×10 ⁹ CFU/ml.

(4) Add formaldehyde solution with a final concentration of 0.2% to the three bacterial cultures and inactivate them at 37°C for 24 hours with continuous stirring at a speed of 100-120 r/min. After inactivation, store them at 2-8°C.

(5) The three antigen solutions were mixed in equal amounts so that the bacterial content of each antigen was 3.0×10 ⁹ CFU/ml. The mixed antigen solution was mixed with the aluminum gel adjuvant at a ratio of 5:1 and stirred at a low speed for 30 minutes to prepare a trivalent inactivated vaccine of Haemophilus parasuis. The number of live bacteria before inactivation of serotype 4 SH strain, serotype 5 GD strain and serotype 12 JS strain in the finished vaccine was 2.5×10 ⁹ CFU/ml.

Example 5

Vaccine guinea pig immune antibody detection test

(1) Immunization, blood collection, and serum separation

Twenty healthy negative guinea pigs (negative for antigens and antibodies of type 4, type 5 and type 12 Haemophilus parasuis) weighing 300-350g were used and divided into two groups, with 10 in each group. Blood was collected and serum was separated. Group 1# was injected intramuscularly with 0.5ml of OMV vaccine (20μg OMV vaccine, type 12 JS1015 strain OMVs prepared in Example 2 were diluted to 40μg/ml with PBS) (0.25ml was injected into each thigh); Group 2# was not immunized as a control, and blood was collected and serum was separated from each guinea pig 21 days after the first immunization, and then a second immunization was carried out using the same method and the same dose, 14 days after the second immunization, and blood was collected from each guinea pig and serum was separated.

(2) Detection of antibody titer

Haemophilus parasuis microagglutination method

Place the serum to be tested in a 56°C water bath and inactivate for 30 minutes. Add 50μ1 sterile PBS solution to each well of the 96-well V-type micro-agglutination plate, and add 50μ1 of the serum to be tested, positive serum and negative serum to each well in the first column, respectively, and blow 8 to 10 times to mix thoroughly. Pipette 50μ1 of the mixed solution from the first column well and add it to the second column well. After mixing, take 50μ1 and add it to the third column well. Perform serial dilutions to the 11th column well in turn, and finally take 50μL of solution from each of the 11th column wells and discard it. Set the 12th column well as the control. Add 50μ1 of Haemophilus parasuis serotype 12 JS strain agglutination antigen to each well in turn; place the micro-agglutination plate on a micro-oscillator and oscillate for about 1 minute, cover and seal the plate after mixing, let it stand at 37°C for 2 hours, then let it stand at 2 to 8°C for 24 hours, observe the agglutination phenomenon and determine the results. The results are shown in Table 1.

The antibodies in the serum of experimental guinea pigs were determined by the microagglutination method of Haemophilus parasuis. The test results showed that specific antibodies could be produced 21 days after immunization with OMV vaccine, and the antibody agglutination titer was ≥1:36. The antibody level continued to increase with the number of immunizations. At 35 days after immunization, the average titer of specific antibodies in the OMV vaccine group reached ≥1:134.

Table 1

Example 6

Vaccine immune challenge protection test

(1) Immunity

25 healthy susceptible piglets (type 4, 5, 12 Haemophilus parasuis antigens and antibodies were all negative) aged 2 to 3 weeks were selected and divided into 5 groups (groups I to V), with 5 pigs in each group. Among them, 2.0 ml of OMV vaccine was injected into the neck muscle of each pig in groups I, II and III (OMVs prepared in Example 2 were diluted to 25 μg/ml with PBS to prepare 50 μg OMV vaccine, and similarly, 100 μg OMV vaccine was prepared by diluting with PBS to 50 μg/ml, and 150 μg OMV vaccine was prepared by diluting with PBS to 75 μg/ml, and the dosage is shown in Table 2). 2.0 ml of whole bacterial vaccine (prepared according to the method described in Example 4) was injected into the neck muscle of each pig in group IV. Group V was used as a control, and each pig was injected with the same dose of sterile saline. After 21 days of immunization, booster immunization was performed once with the same dose and method.

(2) Attack

Haemophilus parasuis serotype 12 strain JS1015 was cultured to the logarithmic growth phase, diluted and counted with PBS, and then injected intraperitoneally with a challenge dose of 5.0×10 ⁹ CFU/pig 14 days after the second immunization.

After the challenge, the incidence and mortality were observed and counted daily. After 14 days, the surviving experimental pigs were killed and autopsied one by one, and the incidence was recorded. After the challenge, at least 4/5 of each control group became ill, and at least 4/5 of each immune group was protected.

Attack results

When the dose of OMVs of Haemophilus parasuis JS strain 12 was 100 μg and 150 μg, the protection rate of group III was 100%, which was equivalent to the protection rate of whole bacterial vaccine of IV (see the table below). In summary, OMVs of Haemophilus parasuis JS strain 12 have a protective effect on animals and can indeed be developed and applied as a new vaccine.

Table 2

The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. The haemophilus parasuis outer membrane vesicle is characterized in that the outer membrane vesicle is secreted by a haemophilus parasuis serotype 12 JS1015 strain, the preservation number of the haemophilus parasuis serotype 12 JS1015 strain is CGMCC No.14418, and the haemophilus parasuis is classified and named as haemophilus parasuis Haemophilus parasuis.

2. The haemophilus parasuis outer membrane vesicle according to claim 1, wherein the outer membrane vesicle particle size is within 200nm and the protein band size is concentrated between 25KDa and 200 KDa.

3. The method for preparing the haemophilus parasuis outer membrane vesicles according to any one of claims 1 to 2, comprising the steps of:

(1) Fermenting and culturing the haemophilus parasuis serotype 12 JS1015 strain to a stable stage to obtain bacterial liquid;

(2) Centrifuging the bacterial liquid at 10000rpm/min and 2-8deg.C for 10min, collecting supernatant, and concentrating with 300kd hollow fiber system to obtain concentrated solution;

(3) PEG with concentration of 7% is added into the concentrated solution, after stirring for 4 hours at 2-8 ℃, standing overnight at 2-8 ℃, then centrifuging for 30min at 12000rpm, and the precipitate is resuspended by PB S and passes through a 0.22 mu m filter membrane to obtain haemophilus parasuis outer membrane vesicles.

4. The method according to claim 3, wherein in the step (1), the fermentation culture is performed by controlling dissolved oxygen to 33-36% by adjusting an intake air amount and a stirring speed; fermenting and culturing for 10-12 h at 37 ℃; the medium for fermentation culture was TSB to which NAD was added at a final concentration of 4. Mu.g/ml and 5% neonatal bovine serum.

5. Use of the outer membrane vesicles prepared according to claims 3-4 for the preparation of a vaccine for the prevention of haemophilus parasuis disease.

6. A vaccine for the prevention of haemophilus parasuis disease comprising the haemophilus parasuis outer membrane vesicle according to any one of claims 1-2.

7. The vaccine for preventing haemophilus parasuis disease according to claim 6, wherein the concentration of haemophilus parasuis outer membrane vesicles in the vaccine is 0.05-20mg/mL.