Preparation method and application of oral vaccine based on bacterial outer membrane vesicles

Abstract

The invention relates to a preparation method and application of an oral vaccine based on bacterial outer membrane vesicles, which can be effectively applied to preparation of drugs for preventing or treating intestinal mucosa pathogen infection and overcome the problems of poor compliance and low mucosa immune response of traditional injection vaccines. The preparation method comprises the following steps: firstly preparing bacterial outer membrane vesicles of alcaligenes faecalis, then preparing DSPE-PEG-antigens, mixing the bacterial outer membrane vesicles and the DSPE-PEG-antigens, stirring to prepare OMV-DSPE-PEG-antigens, dissolving inulin in PBS, heating, oscillating, cooling to room temperature, adding the OMV-DSPE-PEG-antigens, stirring, and standing to obtain the finished oral vaccine. The preparation process is simple, the method is stable and reliable, operation is easy, the production cost is low, the prepared oral vaccine based on the bacterial outer membrane capsule can effectively stimulate immune response of intestinal mucosa and prevent infection of mucosa pathogens, and economic and social benefits are remarkable.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing an oral vaccine based on bacterial outer membrane vesicles and applications thereof. Background Art

[0002] Pathogen infections pose a significant threat to human health, and vaccination is one of the most effective measures to prevent and treat pathogen infections. However, common clinical methods of vaccine administration, such as intramuscular and subcutaneous injections, do not provide mucosal defense capabilities. In comparison, oral vaccines can activate the intestinal mucosal immune response, effectively preventing mucosal infections by pathogens, and exhibit numerous advantages, such as good compliance and high economic benefits. However, the development of oral vaccines faces considerable scientific challenges, such as the complex biological environment of the gastrointestinal tract, which can easily lead to antigen inactivation, and the lack of excellent mucosal immune adjuvants.

[0003] Studies have found that healthy intestinal flora is a key factor in maintaining intestinal immune homeostasis and is crucial to the efficiency of intestinal mucosal immunity. Prebiotics can effectively regulate intestinal flora homeostasis and immune homeostasis, complete the rear support work of the vaccine war, and improve vaccine efficacy. Inulin, as a prebiotic, has been included in the list of food additive safety index ingredients evaluated by the U.S. Food and Drug Administration. After simple processing and treatment, this substance can form a hydrogel, which has a strong adhesion effect on the intestine. In addition, studies have reported that the outer membrane vesicles of Alcaligenes faecalis are a vaccine adjuvant. Based on this, the present invention intends to load the outer membrane vesicles of Alcaligenes faecalis into inulin hydrogel to prepare an oral vaccine to enhance intestinal mucosal immunity, but there has been no public report so far. Summary of the Invention

[0004] In view of the above situation, in order to solve the defects of the existing technology, the purpose of the present invention is to provide a method for preparing an oral vaccine based on bacterial outer membrane vesicles and its application, which can effectively solve the preparation of oral vaccines based on bacterial outer membrane vesicles and overcome the problems of poor compliance and low mucosal immune response of traditional injectable vaccines.

[0005] The technical solution provided by the present invention is a method for preparing an oral vaccine based on bacterial outer membrane vesicles, comprising the following steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: Alcaligenes faecalis suspension was prepared by suspending Bacillus faecalis in distilled water (the same below), adding it to tryptone soy broth medium at an inoculum size of 4%, and aerobically culturing it in a 37°C incubator. The bacterial solution was centrifuged at 6000 g and 4°C for 10-20 min, the precipitate was discarded, and the supernatant was collected; the supernatant was centrifuged again at 7000 g and 4°C for 10-20 min, the supernatant was collected, and filtered through a polycarbonate filter with a pore size of 0.45 μm. The filtrate was centrifuged at 120,000 g and 4°C for 2-3 h, the precipitate was taken, resuspended in PBS (pH 7.4) (the same below), filtered through a polycarbonate filter with a pore size of 0.22 μm, and the filtrate was centrifuged at 120,000 g and 4°C for 2-3 h. h, obtaining a precipitate, i.e., obtaining the bacterial outer membrane vesicles of Alcaligenes faecalis, and then resuspending the precipitate with PBS to obtain a bacterial outer membrane vesicle solution of Alcaligenes faecalis; The tryptone soy broth medium is composed of: 17.0 g tryptone, 3.0 g soy peptone, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, and 2.5 g glucose, and the volume is adjusted to 1 L with water, and the pH value is 7.3±0.2; 2) Preparation of DSPE-PEG-antigen: Dissolve 5-25 mg of antigen protein and 16-80 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 1-5 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 1.3-6.5 mg of 4-dimethylaminopyridine as a catalyst, sonicate at room temperature in the dark for 20-40 min, and stir to react for 60-90 h. After the reaction, centrifuge at 8000 rpm for 10-20 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 rpm for 10-20 min to obtain a precipitate, and wash 2-4 times to obtain DSPE-PEG-antigen. The antigen protein is ovalbumin, hepatitis B surface antigen or Salmonella O-antigen; The molecular weight of the α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) is 2000-6000; 3) Preparation of OMV-DSPE-PEG-antigen: Take 0.5-2.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 20-100 mg of DSPE-PEG-antigen prepared in step 2) and stir in the dark for 6-18 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 0.4-2.0 g of inulin and dissolve it in 1-5 mL of PBS. Oscillate at 70-90°C for 5-10 min. After the solution cools to room temperature, immediately add 20-100 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 2-3 h and let it stand at 4°C for 20-30 h to obtain the finished oral vaccine.

[0006] The invention relates to an application of an oral vaccine based on bacterial outer membrane vesicles prepared by the method in preparing a drug for preventing or treating intestinal mucosal pathogen infection.

[0007] The preparation process of the present invention is simple, the method is stable and reliable, easy to operate, and has low production cost. The prepared oral vaccine based on bacterial outer membrane vesicles can effectively stimulate the intestinal mucosal immune response and prevent infection by mucosal pathogens, opening up a new technical approach to intestinal mucosal immunity. It is an innovation in enhancing intestinal mucosal immune vaccines and has significant economic and social benefits. DETAILED DESCRIPTION

[0008] The specific embodiments of the present invention are further described in detail below with reference to the examples. Example

[0009] The present invention provides a method for preparing an oral vaccine based on bacterial outer membrane vesicles and its application, comprising the following steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: A 4% inoculum of a Bacillus faecalis suspension was added to a tryptone soy broth medium and cultured aerobically in a 37°C incubator. The culture was centrifuged at 6000 g and 4°C for 15 min, the precipitate discarded, and the supernatant collected. The supernatant was centrifuged again at 7000 g and 4°C for 15 min, the supernatant collected, and filtered through a 0.45 μm polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 2.5 h. The precipitate was collected and resuspended in PBS (pH 7.4) and filtered through a 0.22 μm polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 2.5 h to obtain the precipitate, which is the bacterial outer membrane vesicles of Bacillus faecalis. The precipitate was resuspended in PBS to obtain the bacterial outer membrane vesicle solution of Bacillus faecalis. 2) Preparation of DSPE-PEG-antigen: Dissolve 15 mg of antigen protein and 48 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 3 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 3.9 mg of 4-dimethylaminopyridine as a catalyst, and sonicate for 30 min at room temperature in the dark. Stir and react for 75 h. After the reaction, centrifuge at 8000 r / min for 15 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 15 min to obtain a precipitate, and wash three times to obtain DSPE-PEG-antigen. 3) Preparation of OMV-DSPE-PEG-antigen: Take 1.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 60 mg of the DSPE-PEG-antigen prepared in step 2) and stir in the dark for 12 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 1.2 g of inulin and dissolve it in 3 mL of PBS. Oscillate at 80°C for 8 min. After the solution cools to room temperature, immediately add 20-100 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 2.5 h and let it stand at 4°C for 25 h to obtain the finished oral vaccine. Example

[0010] The present invention provides a method for preparing an oral vaccine based on bacterial outer membrane vesicles and its application, comprising the following steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: A 4% inoculum of a suspension of Alcaligenes faecalis was added to a tryptone soy broth medium and cultured aerobically in a 37°C incubator. The culture was centrifuged at 6000 g and 4°C for 10 min, the precipitate discarded, and the supernatant collected. The supernatant was centrifuged again at 7000 g and 4°C for 10 min, the supernatant collected, and filtered through a 0.45 μm polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 2 h. The precipitate was collected, resuspended in PBS (pH 7.4), and filtered through a 0.22 μm polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 2 h to obtain the precipitate, which is the bacterial outer membrane vesicles of Alcaligenes faecalis. The precipitate was then resuspended in PBS to obtain the bacterial outer membrane vesicle solution of Alcaligenes faecalis. 2) Preparation of DSPE-PEG-antigen: Dissolve 5 mg of antigen protein and 16 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 1 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 1.3 mg of 4-dimethylaminopyridine as a catalyst, sonicate for 20 min at room temperature in the dark, and then stir to react for 60 h. After the reaction, centrifuge at 8000 r / min for 10-20 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 10 min to obtain a precipitate, and wash twice to obtain DSPE-PEG-antigen. 3) Preparation of OMV-DSPE-PEG-antigen: Take 0.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 20 mg of DSPE-PEG-antigen prepared in step 2) and stir in the dark for 6 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 0.4 g of inulin and dissolve it in 1.0 mL of PBS. Oscillate at 70°C for 5 min. After the solution cools to room temperature, immediately add 20 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 2 h and let it stand at 4°C for 20 h to obtain the finished oral vaccine. Example

[0011] The present invention provides a method for preparing an oral vaccine based on bacterial outer membrane vesicles and its application, comprising the following steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: A 4% inoculum of a Bacillus faecalis suspension was added to a tryptone soy broth medium and cultured aerobically in a 37°C incubator. The culture was centrifuged at 6000 g and 4°C for 20 min, the precipitate discarded, and the supernatant collected. The supernatant was centrifuged again at 7000 g and 4°C for 20 min, the supernatant collected, and filtered through a 0.45 μm polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 3 h. The precipitate was collected and resuspended in PBS (pH 7.4) and filtered through a 0.22 μm polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 3 h to obtain the precipitate, which is the bacterial outer membrane vesicles of Bacillus faecalis. The precipitate was resuspended in PBS to obtain the bacterial outer membrane vesicle solution of Bacillus faecalis. 2) Preparation of DSPE-PEG-antigen: Dissolve 25 mg of antigen protein and 80 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 5 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 6.5 mg of 4-dimethylaminopyridine as a catalyst, and sonicate for 40 min at room temperature in the dark. Stir and react for 90 h. After the reaction, centrifuge at 8000 r / min for 20 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 20 min to obtain a precipitate, and wash four times to obtain DSPE-PEG-antigen. 3) Preparation of OMV-DSPE-PEG-antigen: Take 2.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 100 mg of DSPE-PEG-antigen prepared in step 2) and stir in the dark for 18 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 2.0 g of inulin and dissolve it in 5 mL of PBS. Oscillate at 90°C for 10 min. After the solution cools to room temperature, immediately add 100 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 3 h and let it stand at 4°C for 30 h to obtain the finished oral vaccine.

[0012] The invention relates to an application of an oral vaccine based on bacterial outer membrane vesicles prepared by the method in preparing a drug for preventing or treating intestinal mucosal pathogen infection.

[0013] The present invention inserts the hydrophobic end of the DSPE-PEG-antigen into the phospholipid bilayer of the outer membrane vesicles (OMV) of Alcaligenes faecalis through hydrophobic interactions, and uses inulin hydrogel to protect the OMV-DSPE-PEG-antigen to complete the construction of an oral vaccine. After oral administration, inulin can regulate the homeostasis of intestinal flora and improve vaccine efficacy. The inulin hydrogel system protects the antigen from the harsh gastrointestinal environment and adheres to the intestine, prolonging its residence time in the intestine. As an excellent adjuvant-assisted vaccine, OMV effectively activates the body's mucosal immune response and promotes the production of intestinal IgA. Scientific experiments have shown that it can significantly enhance intestinal mucosal immunity, achieving very satisfactory technical results. The specific experimental data are as follows (taking Example 1 as an example): Characterization of Oral Vaccines Based on Bacterial Outer Membrane Vesicles:

[0014] 1. Scanning electron microscopy of oral vaccines An appropriate amount of the oral vaccine was freeze-dried, dehydrated, and then gold-sprayed and observed under a scanning electron microscope. The results showed that the oral hydrogel vaccine had a rough surface and a large number of particles attached to the interior, indicating that the OMV-DSPE-PEG-antigen particles were loaded into the hydrogel, confirming the successful construction of the oral vaccine.

[0015] 2. Stability study of oral vaccines in gastric and intestinal fluids Alcaligenes faecalis outer membrane vesicles were labeled with a green fluorescent cell membrane dye, while Salmonella O-antigen was labeled with rhodamine B. The experiment was divided into three groups: a control group (oral vaccine group), a gastric fluid group (oral vaccine group incubated in gastric fluid for 2 hours), and an intestinal fluid group (oral vaccine group incubated in intestinal fluid for 6 hours). Under dark conditions, 10-20 μL of the vaccine suspension from each group was aspirated onto a glass slide, covered with a coverslip, and sealed with nail polish on all sides. The slides were then observed under a laser confocal microscope. Results showed that after 2 hours of treatment with gastric fluid and 6 hours of treatment with intestinal fluid, the green fluorescence of the bacterial outer membrane vesicles labeled with the green fluorescent cell membrane dye and the red fluorescence of the rhodamine B-labeled antigen still colocalized, consistent with the results in the control group. These results demonstrate that the vaccine, protected by the inulin hydrogel, can remain stable in the harsh physiological environment of the gastrointestinal tract. Experiment on the effect of oral vaccination on the level of secretory immunoglobulin A in mice:

[0016] The experiment was divided into three groups: a blank group (phosphate buffer saline), a control group (Salmonella O-antigen group), and an experimental group (oral vaccine group). Healthy C57BL / 6 mice aged 6-8 weeks were selected and administered with normal saline, Salmonella O-antigen, and oral vaccine, respectively, using a 1 mL syringe on days 0, 7, and 14. After the third dose, feces were collected from each group and homogenized at a ratio of 1 g feces to 9 mL PBS. The homogenization was centrifuged at 5000 rpm for 15 minutes, and the supernatant was collected and assayed for secretory immunoglobulin A (SIgA) levels using an ELISA kit. Furthermore, the mice were euthanized, dissected, and their intestines flushed with an appropriate amount of PBS. The flushing fluid was collected and centrifuged at 5000 rpm for 15 minutes, and the supernatant was used to assay for SIGA levels in each group. The levels of secretory immunoglobulin A (SIgA) in the feces of mice in the oral vaccination group were 4.7 times and 2.4 times higher than those in the saline group and the Salmonella O-antigen group, respectively. The levels of SgA in the intestinal lavage fluid of mice in the oral vaccination group were 4.5 times and 2.9 times higher than those in the saline group and the Salmonella O-antigen group, respectively. These results indicate that oral vaccination significantly increases the levels of SgA produced by mice. Pharmacodynamics experiments on oral vaccines to prevent Salmonella infection:

[0017] The experiment was divided into three groups: a blank group (phosphate buffer solution), a control group (Salmonella O-antigen group), and an experimental group (oral vaccine group). Six- to eight-week-old healthy C57BL / 6 mice were orally administered with normal saline, Salmonella O-antigen, and oral vaccine using a 1 mL syringe on days 0, 7, and 14, respectively. On days 21, 22, and 23, the mice were gavage-administered with Salmonella typhimurium. Body weight, diarrhea, and activity were recorded daily. Feces were collected from all groups on day 27 for 16S rDNA high-throughput sequencing. Results showed that mice in the normal saline group experienced continuous weight loss and severe diarrhea, while mice in the oral vaccine group had milder symptoms that gradually improved. High-throughput 16S rDNA sequencing revealed that the fecal counts of Salmonella in mice receiving the oral vaccine were significantly lower than those in the other groups, and by day 27, the microbial composition approached that of healthy mice, demonstrating the effectiveness of the oral vaccine in preventing Salmonella infection.

[0018] After repeated tests, the same or similar results were obtained. At the same time, the same experiments were carried out on other embodiments, and the same or similar test results were obtained. They will not be repeated here.

[0019] Tests have shown that the method of the present invention is stable and reliable, and the resulting product can effectively solve the problems of poor compliance and low mucosal immune response of traditional injectable vaccines. Compared with existing technologies, it has the following outstanding beneficial technical effects: Inulin hydrogel protects antigens from passing through the stomach smoothly and avoiding inactivation in the gastrointestinal environment; Under the combined action of antigenic proteins and Alcaligenes faecalis outer membrane vesicle adjuvant, this oral vaccine effectively stimulates the intestinal mucosal immune response, promotes intestinal secretory immunoglobulin A, and enhances intestinal mucosal immunity; Inulin prebiotics can regulate intestinal flora to maintain intestinal immune homeostasis and enhance vaccine efficacy.

[0020] In summary, the preparation method of the present invention is easy to operate, stable and reliable, and has a simple preparation process. Compared with traditional injectable vaccines, its product has the advantages of convenient administration, good compliance, and avoidance of cross-contamination. It effectively solves the problem that traditional injectable vaccines are difficult to activate mucosal immune responses. It is an innovation in oral vaccines and has significant economic and social benefits.

[0021] It should be pointed out that the above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any technician familiar with this profession can make changes or modify the technical content disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention, and all of them fall within the scope of protection of the present invention.

Claims

1. A method for preparing an oral vaccine based on bacterial outer membrane vesicles, characterized in that: The specific steps are as follows: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: Alcaligenes faecalis suspension was prepared by suspending the culture medium in distilled water, adding the suspension to a 4% inoculum into tryptone soy broth medium, and aerobically culturing the suspension in a 37°C incubator. The suspension was centrifuged at 6000 g and 4°C for 10-20 min, the precipitate was discarded, and the supernatant was collected. The supernatant was centrifuged again at 7000 g and 4°C for 10-20 min, the supernatant was collected, and filtered through a 0.45 μm pore size polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 2-3 h, the precipitate was collected, resuspended in PBS (pH 7.4), and filtered through a 0.22 μm pore size polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 2-3 h to obtain a precipitate, namely, bacterial outer membrane vesicles of Alcaligenes faecalis. The bacterial outer membrane vesicle solution of Alcaligenes faecalis was resuspended in PBS. The tryptone soy broth medium is composed of: 17.0 g tryptone, 3.0 g soy peptone, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, and 2.5 g glucose, and the volume is adjusted to 1 L with water, and the pH value is 7.3±0.2; 2) Preparation of DSPE-PEG-antigen: Dissolve 5-25 mg of antigen protein and 16-80 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 1-5 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 1.3-6.5 mg of 4-dimethylaminopyridine as a catalyst, sonicate at room temperature in the dark for 20-40 min, and stir to react for 60-90 h. After the reaction is completed, centrifuge at 8000 r / min for 10-20 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 10-20 min to obtain a precipitate, and wash 2-4 times to obtain DSPE-PEG-antigen. The antigen protein is ovalbumin, hepatitis B surface antigen or Salmonella O-antigen; The molecular weight of the α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) is 2000-6000; 3) Preparation of OMV-DSPE-PEG-antigen: Take 0.5-2.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 20-100 mg of DSPE-PEG-antigen prepared in step 2) and stir in the dark for 6-18 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 0.4-2.0 g of inulin and dissolve it in 1-5 mL of PBS. Oscillate at 70-90°C for 5-10 min. After the solution cools to room temperature, immediately add 20-100 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 2-3 h and let it stand at 4°C for 20-30 h to obtain the finished oral vaccine.

2. The method for preparing an oral vaccine based on bacterial outer membrane vesicles according to claim 1, characterized in that: Here are the steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: A 4% inoculum of a Bacillus faecalis suspension was added to a tryptone soy broth medium and cultured aerobically in a 37°C incubator. The bacterial suspension was centrifuged at 6000 g and 4°C for 15 min, the precipitate was discarded, and the supernatant was collected. The supernatant was centrifuged again at 7000 g and 4°C for 15 min, the supernatant was collected, and filtered through a 0.45 μm polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 2.5 h. The precipitate was collected and resuspended in PBS (pH 7.4) and filtered through a 0.22 μm polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 2.5 h to obtain the precipitate, which is the bacterial outer membrane vesicles of Bacillus faecalis. The precipitate was resuspended in PBS to obtain the bacterial outer membrane vesicle solution of Bacillus faecalis. 2) Preparation of DSPE-PEG-antigen: Dissolve 15 mg of antigen protein and 48 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 3 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 3.9 mg of 4-dimethylaminopyridine as a catalyst, and sonicate for 30 min at room temperature in the dark. Stir and react for 75 h. After the reaction, centrifuge at 8000 r / min for 15 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 15 min to obtain a precipitate, and wash three times to obtain DSPE-PEG-antigen. 3) Preparation of OMV-DSPE-PEG-antigen: Take 1.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 60 mg of the DSPE-PEG-antigen prepared in step 2) and stir in the dark for 12 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 1.2 g of inulin and dissolve it in 3 mL of PBS. Oscillate at 80°C for 8 min. After the solution cools to room temperature, immediately add 20-100 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 2.5 h and let it stand at 4°C for 25 h to obtain the finished oral vaccine.

3. The method for preparing an oral vaccine based on bacterial outer membrane vesicles according to claim 1, characterized in that: Here are the steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: A 4% inoculum of a Bacillus faecalis suspension was added to a tryptone soy broth medium and cultured aerobically in a 37°C incubator. The bacterial suspension was centrifuged at 6000 g and 4°C for 10 min, the precipitate was discarded, and the supernatant was collected. The supernatant was centrifuged again at 7000 g and 4°C for 10 min, the supernatant was collected, and filtered through a 0.45 μm polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 2 h. The precipitate was collected and resuspended in PBS (pH 7.4) and filtered through a 0.22 μm polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 2 h to obtain the precipitate, which is the bacterial outer membrane vesicles of Bacillus faecalis. The precipitate was resuspended in PBS to obtain the bacterial outer membrane vesicle solution of Bacillus faecalis. 2) Preparation of DSPE-PEG-antigen: Dissolve 5 mg of antigen protein and 16 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 1 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 1.3 mg of 4-dimethylaminopyridine as a catalyst, sonicate for 20 min at room temperature in the dark, and then stir to react for 60 h. After the reaction, centrifuge at 8000 r / min for 10-20 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 10 min to obtain a precipitate, and wash twice to obtain DSPE-PEG-antigen. 3) Preparation of OMV-DSPE-PEG-antigen: Take 0.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 20 mg of DSPE-PEG-antigen prepared in step 2) and stir in the dark for 6 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 0.4 g of inulin and dissolve it in 1.0 mL of PBS. Oscillate at 70°C for 5 min. After the solution cools to room temperature, immediately add 20 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 2 h and let it stand at 4°C for 20 h to obtain the finished oral vaccine.

4. The method for preparing an oral vaccine based on bacterial outer membrane vesicles according to claim 1, characterized in that: Here are the steps: 1) Preparation of bacterial outer membrane vesicles of Alcaligenes faecalis: A 4% inoculum of a Bacillus faecalis suspension was added to a tryptone soy broth medium and cultured aerobically in a 37°C incubator. The culture was centrifuged at 6000 g and 4°C for 20 min, the precipitate discarded, and the supernatant collected. The supernatant was centrifuged again at 7000 g and 4°C for 20 min, the supernatant collected, and filtered through a 0.45 μm polycarbonate filter. The filtrate was centrifuged at 120,000 g and 4°C for 3 h. The precipitate was collected and resuspended in PBS (pH 7.4) and filtered through a 0.22 μm polycarbonate filter to obtain a filtrate. The filtrate was centrifuged again at 120,000 g and 4°C for 3 h to obtain the precipitate, which is the bacterial outer membrane vesicles of Bacillus faecalis. The precipitate was resuspended in PBS to obtain the bacterial outer membrane vesicle solution of Bacillus faecalis. 2) Preparation of DSPE-PEG-antigen: Dissolve 25 mg of antigen protein and 80 mg of α-propionaldehyde-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE-PEG-CHO) in 5 mL of dimethyl sulfoxide, respectively. Mix the two solutions, add 6.5 mg of 4-dimethylaminopyridine as a catalyst, and sonicate for 40 min at room temperature in the dark. Stir and react for 90 h. After the reaction, centrifuge at 8000 r / min for 20 min to obtain a precipitate. Add sterile water, centrifuge again at 8000 r / min for 20 min to obtain a precipitate, and wash four times to obtain DSPE-PEG-antigen. 3) Preparation of OMV-DSPE-PEG-antigen: Take 2.5 mL of the bacterial outer membrane vesicle solution of Alcaligenes faecalis prepared in step 1) and mix it with 100 mg of DSPE-PEG-antigen prepared in step 2) and stir in the dark for 18 h to obtain OMV-DSPE-PEG-antigen; 4) Preparation of oral vaccines Weigh 2.0 g of inulin and dissolve it in 5 mL of PBS. Oscillate at 90°C for 10 min. After the solution cools to room temperature, immediately add 100 mg of the OMV-DSPE-PEG-antigen prepared in step 3). Stir for 3 h and let it stand at 4°C for 30 h to obtain the finished oral vaccine.

5. Use of an oral vaccine based on bacterial outer membrane vesicles prepared by the method according to any one of claims 1 to 4 in the preparation of a medicament for preventing or treating intestinal mucosal pathogen infection.