A nano-vaccine based on Helicobacter pylori outer membrane vesicles, its preparation method and application

Abstract

The present invention belongs to the field of vaccine preparation, and specifically relates to a nano-vaccine based on Helicobacter pylori outer membrane vesicles, and a preparation method and application thereof. In the present invention, OMV and LPS of Helicobacter pylori are used as antigens and adjuvants, and DMON is used as a carrier. Then, electrostatic interaction is utilized to enable DMON to adsorb LPS small molecules, and a liposome extruder is used to uniformly wrap Helicobacter pylori OMV on the surface of the DMON nanomaterial, thereby obtaining the nano-vaccine based on Helicobacter pylori outer membrane vesicles. The present invention proves through in vitro and in vivo experiments that the nano-vaccine can be taken up by macrophages, promote the phagocytosis ability of macrophages, significantly increase the levels of various immune cytokines secreted by macrophages, and can induce high-level antigen-specific humoral immunity, mucosal immunity, and Th1 / Th2 / Th17-type cellular immune responses in mice.

Description

Technical Field

[0001] The present invention belongs to the field of vaccine preparation, and particularly relates to a nano-vaccine based on Helicobacter pylori outer membrane vesicles, a preparation method thereof, and an application thereof. Background Art

[0002] Helicobacter pylori is a microaerophilic bacterium with a single polar and multiple flagella, spiral-shaped, and Gram-negative. It can move in the gastric mucus layer, secrete urease to decompose urea to produce ammonia, and adapt to the acidic gastric environment. Most Helicobacter pylori infections are usually asymptomatic, but this condition persists as a chronic infection and increases the risk of peptic ulcer, chronic gastritis, and gastric cancer. Currently, antibiotic treatment is the most commonly used regimen for treating Helicobacter pylori infection. Clinically, triple or quadruple therapy is generally used. However, with the popularization of antibiotic treatment, antibiotic resistance has become another prominent clinical problem. Developing safe and effective vaccines has become an important measure for preventing Helicobacter pylori infection.

[0003] Outer membrane vesicles (OMVs) are spherical nanostructures with a diameter ranging from 20 to 250 nm secreted by Gram-negative bacteria, containing bacterial components such as proteins, DNA, RNA, lipopolysaccharide (LPS), enzymes, and peptidoglycan. OMVs can effectively induce innate and adaptive immune responses against bacterial infections in the body, making them potential new vaccine candidates. OMVs cannot self-replicate, solving the main safety problems in the application of traditional inactivated whole-bacteria vaccines. Their nano-size is conducive to the uptake by antigen-presenting cells.

[0004] Currently, more and more studies have focused on using nanomaterials to treat drug-resistant bacterial infections. Compared with pure antigen vaccines, the advantages of nanomaterial vaccines mainly include: (1) Nanocarriers can avoid the rapid degradation of antigens and improve the stability of vaccine preparation; (2) Provide good adjuvant properties and promote the activation of antigen-presenting cells; (3) The nano-scale enhances the accumulation of antigens in lymph nodes and further enhances their immune response ability. Summary of the Invention

[0005] In order to overcome the deficiencies and drawbacks of the prior art, the primary object of the present invention is to provide a preparation method of a nano-vaccine based on Helicobacter pylori outer membrane vesicles. This method uses Helicobacter pylori outer membrane vesicles (OMVs) as antigens, lipopolysaccharide (LPS) as an adjuvant, and dendritic mesoporous organosilica (DMON) as a carrier. The electrostatic interaction is used to make DMON adsorb LPS small molecules; finally, a liposome extruder is used to uniformly wrap OMVs on the surface of DMON nanomaterials to obtain a nano-vaccine based on Helicobacter pylori outer membrane vesicles.

[0006] Another object of the present invention is to provide a nano-vaccine based on Helicobacter pylori outer membrane vesicles prepared by the above preparation method. This nano-vaccine can be taken up by macrophages, promote the phagocytic ability of macrophages, significantly increase the levels of various immune cytokines secreted by macrophages, and can induce high levels of antigen-specific humoral immunity, mucosal immunity, and Th1 / Th2 / Th17 cell immune responses in mice.

[0007] Another object of the present invention is to provide the application of the above nano-vaccine based on Helicobacter pylori outer membrane vesicles.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] A preparation method of a nano-vaccine based on Helicobacter pylori outer membrane vesicles, comprising the following steps:

[0010] (1) Isolate and purify OMV

[0011] Take a Helicobacter pylori bacterial suspension, centrifuge at a low speed of 8000 - 10000 rpm to obtain a supernatant and a precipitate respectively; filter the supernatant and take the filtrate; ultra-centrifuge the filtrate at 100000 - 300000 g, discard the supernatant, and resuspend the precipitate to obtain an OMV solution;

[0012] (2) Extract LPS

[0013] Take the precipitate obtained by low-speed centrifugation in step (1), and extract LPS by referring to the conventional method to obtain an LPS solution;

[0014] (3) Synthesize DMON

[0015] Using cetyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) as template agents, tetraethyl orthosilicate (TEOS) and 1,2-bis(triethoxysilyl)ethane (BTEE) as silica sources, and triethanolamine (TEA) as a catalyst, DMON is prepared by a one-pot method;

[0016] (4) Prepare DMON-NH 2

[0017] Mix the DMON prepared in step (3) with ethanol to obtain a DMON ethanol solution; mix the DMON ethanol solution with 3-aminopropyltriethoxysilane (APTES) for an amination reaction. After the reaction is completed, purify and dry the reaction product to obtain DMON-NH 2 ;

[0018] (5) Prepare LPS@DMON@OMV

[0019] Mix the DMON-NH prepared in step (4)2 Mix with water to obtain DMON-NH 2 solution; Mix the LPS solution prepared in step (2) with the DMON-NH 2 solution, stir and react; After the reaction is completed, purify the reaction product and resuspend it with a buffer solution to obtain the LPS@DMON solution; Mix the LPS@DMON solution with the OMV solution prepared in step (1), and extrude it 10-20 times using a liposome extruder, and finally centrifuge to remove the unloaded OMV to obtain a nano-vaccine based on Helicobacter pylori outer membrane vesicles;

[0020] The Helicobacter pylori bacterial suspension described in step (1) is preferably prepared by the following method:

[0021] Place the frozen Helicobacter pylori SS1 strain on Columbia blood agar medium, culture it at 37°C in a microaerobic environment for 48-72 h, then collect the colonies and add them to a liquid medium for expanded culture, and continue to culture for 48-72 h;

[0022] The specific conditions of the microaerobic environment are preferably 5% O 2 、10% CO 2 、85% N 2 ;

[0023] The OD 600nm of the Helicobacter pylori bacterial suspension described in step (1) is preferably 1-2;

[0024] The conditions of the low-speed centrifugation described in step (1) are preferably: low-speed centrifugation at 4°C, 8000-10000 rpm for 2-3 times, 10-20 min each time. After each centrifugation, take the supernatant, and centrifuge the supernatant again; The supernatant obtained from the last centrifugation is used for subsequent filtration, and the precipitates after each centrifugation are combined for subsequent extraction of LPS;

[0025] The filtration described in step (1) is preferably carried out 2-3 times using a 0.22 μm microporous filter;

[0026] The conditions of the ultracentrifugation described in step (1) are preferably ultracentrifugation at 4°C, 100000-300000 g for 1.5-2.5 h;

[0027] The resuspension described in step (1) is preferably carried out using PBS buffer solution;

[0028] The preparation method of DMON described in step (3) specifically includes the following steps:

[0029] ①Under stirring conditions, add TEA to water and stir in an oil bath at 70 - 90 °C for 0.3 - 0.7 h; add CTAB and NaSal to the above solution and continue stirring in an oil bath at 70 - 90 °C for 0.8 - 1.2 h to fully dissolve CTAB and NaSal and form a stable template structure;

[0030] ②Mix TEOS and BTEE and then add them to the system in step (1), and continue stirring in an oil bath at 70 - 90 °C for 0.5 - 1.5 h; after the reaction is completed, purify and dry the reaction product to obtain DMON;

[0031] The mass ratio of TEA, CTAB and NaSal described in step ① is preferably (60 - 80):(350 - 400):(150 - 180);

[0032] The volume ratio of TEOS and BTEE described in step ② is preferably (1.5 - 2.5):(1.4 - 1.8);

[0033] The mass - volume ratio of TEA described in step ① and TEOS described in step ② in mg:mL is preferably (60 - 80):(1.5 - 2.5);

[0034] The specific operation of the purification described in step ② is preferably:

[0035] Reflux - extract the reaction product obtained in step ② with a mixed solution of hydrochloric acid and methanol; the volume ratio of hydrochloric acid and methanol is (1:8)-(1:10); the conditions of the reflux - extraction are preferably: temperature 55 - 65 °C, repeat extraction 2 - 4 times, and the extraction time for each time is 5 - 7 h to ensure complete removal of the template agent (structure - directing agent), etc.;

[0036] The drying conditions described in step ② are preferably vacuum drying for 12 - 24 h;

[0037] In the DMON ethanol solution described in step (4), the mass - volume ratio of DMON to APTES in mg:μL is 10:1, and the concentration of the DMON ethanol solution is preferably 5 - 20 mg / mL;

[0038] The conditions of the amination reaction described in step (4) are preferably stirring at 300 - 600 rpm for 12 - 24 h to ensure full progress of the reaction;

[0039] The specific operations of the purification and drying described in step (4) are preferably:

[0040] After the reaction is completed, perform solid - liquid separation, wash with ethanol or water to remove unreacted APTES, and vacuum - dry the washed product for 12 - 24 h to obtain DMON - NH 2 ;

[0041] The mass ratio of LPS in the LPS solution, OMV in the OMV solution, and DMON-NH in the DMON-NH 2 solution in the DMON-NH 2 solution is preferably 0.75:(20 - 40):200;

[0042] The volume ratio of the LPS solution to the DMON-NH 2 solution described in step (5) is preferably (1:4)-(1:5);

[0043] The mass ratio of LPS in the LPS solution to DMON-NH 2 in the DMON-NH 2 solution in the solution is preferably 0.075:20;

[0044] The concentration of the LPS solution described in step (5) is preferably 150 μg / mL;

[0045] The volume ratio of the LPS@DMON solution to the OMV solution described in step (5) is preferably 1:1;

[0046] DMON-NH in the LPS@DMON solution described in step (5) 2 and OMV in the OMV solution have a mass ratio preferably of 5:0.5;

[0047] The concentration of the OMV solution described in step (5) is preferably 1mg / mL;

[0048] The conditions of the reaction described in step (5) are preferably stirring at 300 - 600 rpm for 12 - 24 h;

[0049] The purification conditions described in step (5) are preferably:

[0050] After the reaction is completed, solid-liquid separation is carried out, washed with water to remove unreacted LPS, and the washed product is resuspended with PBS buffer solution;

[0051] When the LPS@DMON solution and the OMV solution are mixed and extruded in step (5), it is preferably carried out at 4°C;

[0052] The centrifugation conditions described in step (5) are preferably centrifugation at 4°C, 7000 - 10000 rpm for 10 - 20 min;

[0053] A nano-vaccine based on Helicobacter pylori outer membrane vesicles is prepared by the above preparation method;

[0054] Use of the described nano-vaccine based on Helicobacter pylori outer membrane vesicles in the preparation of products for preventing and treating peptic ulcer, chronic gastritis and gastric cancer;

[0055] Technical principle of the present invention:

[0056] The present invention provides a nano-vaccine based on Helicobacter pylori outer membrane vesicles. This vaccine uses Helicobacter pylori OMV as an antigen, LPS as an adjuvant, and DMON as a carrier. Among them, LPS as an adjuvant can improve the immunogenicity of OMV and enhance the immune effect. DMON has a multi-level nanoporous and dendritic structure, enabling it to have a larger specific surface area to display antigens and adsorb adjuvant small molecules.

[0057] Since LPS and DMON are negatively charged, the present invention first uses APTES (3-aminopropyltriethoxysilane) to amino-modify DMON to make it positively charged; then DMON-NH 2 Loads LPS, which belongs to a small molecule structure, through electrostatic interaction, enabling it to adsorb to the pores and surface of DMON to form LPS@DMON; then further mixes and extrudes the antigen OMV and LPS@DMON. The antigen OMV will encapsulate LPS@DMON to form a stable circular nanoparticle vaccine. If OMV and DMON are first mixed, OMV will encapsulate DMON and form a film on its surface, then LPS cannot be loaded onto the entire nanoparticle but is dispersed in the solution. After subsequent centrifugation, LPS is not included in the final product obtained. In addition, the present invention selects OMV to encapsulate LPS@DMON because OMV has certain natural biocompatibility and targeting properties and is easily actively taken up by antigen-presenting cells, so other components are encapsulated into OMV.

[0058] In addition, the dosage relationship among LPS, OMV, and DMON is very important. The ratio provided by the present invention can obtain a relatively stable protein and LPS loading ratio. Excessive use of LPS will increase toxicity, while too little will not have enough effect; excessive use of OMV is a waste, while too little will result in a low protein concentration; excessive use of DMON is not good for passing through the extruder. An appropriate dosage ratio can improve safety and avoid wasting raw materials.

[0059] The present invention has the following advantages and effects compared with the prior art:

[0060] (1) Multiple components work synergistically to break through the limitation of insufficient immunogenicity of a single antigen. The present invention uses Helicobacter pylori natural OMV as the core antigen, retaining multiple antigen epitopes of the pathogen (such as outer membrane proteins, lipoproteins, etc.). Compared with traditional single recombinant protein antigens, it can induce more comprehensive humoral immunity and cellular immunity. Verified by animal experiments, the serum specific IgG antibody titer after immunization with this vaccine can reach 1:12800, and significantly activates the Th1 / Th2 / Th17 mixed immune response.

[0061] (2) The present invention uses Helicobacter pylori homologous lipopolysaccharide (LPS) as a natural adjuvant, avoiding the toxicity risks of exogenous adjuvants (such as aluminum salts, CpG, etc.).

[0062] (3) The preparation process of the present invention is simple and suitable for large-scale production, avoiding the complex chemical cross-linking or genetic engineering modification steps of traditional vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 are transmission electron microscope images of OMV, DMON, and LPS@DMON@OMV, where A: OMV, B: DMON, C: LPS@DMON@OMV.

[0064] Figure 2 is the analysis chart of the surface Zeta potential results of each component.

[0065] Figure 3 is the SDS-PAGE result chart of the protein components of OMV, DMON, DMON@OMV, and LPS@DMON@OMV.

[0066] Figure 4 is the analysis chart of the results of the cytotoxicity detection of LPS@DMON@OMV on macrophages.

[0067] Figure 5 is the analysis chart of the results of macrophage uptake of OMV and LPS@DMON@OMV.

[0068] Figure 6 is the analysis chart of the results of the secretion of immune cytokines after macrophages are treated with OMV, DMON@OMV, and LPS@DMON@OMV respectively.

[0069] Figure 7 is the analysis chart of the anti-Helicobacter pylori antibody titer curve in the serum of immunized mice.

[0070] Figure 8 is the analysis chart of the results of the specificity detection of anti-Helicobacter pylori antibodies in the serum of immunized mice.

[0071] Figure 9 is the analysis chart of the IgA levels in the vaginal secretion supernatant and gastric homogenate supernatant, where A: vaginal secretion supernatant, B: gastric homogenate supernatant.

[0072] Figure 10 is the analysis chart of the results of splenocyte immune factor analysis.

[0073] Figure 11It is a result analysis chart of the effects of post-immunization in routine blood analysis on immune cells in the blood of each group (white blood cells WBC, lymphocytes LYM, neutrophils NEU, and monocytes MON).

[0074] Figure 12 It is a result analysis chart of the effects of post-immunization in routine blood analysis on the content of red blood cells (RBC), hemoglobin content (HGB), and platelet content (PLT) in the blood of each group.

[0075] Figure 13 It is a HE staining result chart of the heart, liver, spleen, lungs, and kidneys of mice in each group collected by microscope. Specific implementation manners

[0076] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0077] The Helicobacter pylori in the embodiment is the Helicobacter pylori SS1 strain, which has been disclosed in the reference (Sun Jingguo, Zhu Wenshuai, Lu Yi, etc. Effects of Helicobacter pylori infection on the m6A level in gastric cancer cells and its mechanism [J]. Journal of Shandong University (Health Sciences), 2023, 61(09): 10-18.).

[0078] Preparation of Columbia blood agar medium in the embodiment: Columbia agar medium (purchased from Guangdong Huankai Microbiological Technology Co., Ltd.), after being configured and sterilized according to the usage method, 10% sterile defibrinated sheep blood is added to obtain Columbia blood agar medium.

[0079] Preparation of exosome-free fetal bovine serum in the embodiment: The Sijiqing premium fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.) is centrifuged at 4°C and 100,000g for 12h, and the supernatant is taken to obtain it.

[0080] Preparation of Brucella broth medium in the embodiment: It is obtained by configuring the purchased Brucella broth dry powder medium (Guangdong Huankai Microbiological Technology Co., Ltd.) according to the usage method.

[0081] DMEM high-glucose medium is purchased from Gibco (product number: C11995500BT).

[0082] DMEM complete medium: 90% DMEM + 10% fetal bovine serum (Gibco, product number C0235), and additionally 1% penicillin-streptomycin double antibody (Gibco, product number 15140122) is added.

[0083] Example 1 Preparation of the nano-vaccine LPS@DMON@OMV based on Helicobacter pylori outer membrane vesicles

[0084] (1) Liquid culture of Helicobacter pylori and isolation and purification of OMV

[0085] ① Liquid culture of Helicobacter pylori

[0086] Take out the frozen Helicobacter pylori SS1 strain from the -80°C refrigerator, thaw it on ice, pipette 100 μL of the bacterial solution and drop it onto the freshly prepared Columbia blood agar medium, and spread it evenly with a sterile spreading rod. Incubate it upside down in an environment of 37°C and microaerobic (5% O 2 , 10% CO 2 , 85% N 2 ) for 48 h; then collect the colonies on the solid plate with sterile 1×PBS buffer, take 500 μL of the bacterial solution with OD 600nm = 1.5 and place it in a bacterial culture dish containing 10 mL of Brucella broth medium supplemented with 10% (v / v) exosome-free fetal bovine serum, and continue to culture for 72 h;

[0087] ② Isolation and purification of OMV

[0088] Collect the Helicobacter pylori bacterial suspension (OD 600nm = 1 - 2) cultured in liquid in step ①, centrifuge at low speed at 4°C and 10000 rpm for 2 times, each time for 20 min. Take the supernatant after each centrifugation, and centrifuge the supernatant again; collect the supernatant obtained from the last centrifugation. The precipitates after each centrifugation are combined and used for subsequent extraction of LPS; the supernatant obtained from the last centrifugation is filtered 2 times with a 0.22 μm microporous filter, and take the filtrate; the filtrate is ultracentrifuged at 4°C and 200000 g for 2 h, discard the supernatant, resuspend the precipitate with sterile 1×PBS buffer, and store it at -80°C;

[0089] (2) Extraction of LPS

[0090] Take the precipitate obtained by low-speed centrifugation in step (1), and extract LPS (the LPS here is mainly the LPS on Helicobacter pylori and part of the LPS in OMV) using a bacterial lipopolysaccharide extraction kit (Boster Biological Technology, product number: BB-31302). The specific method is as follows:

[0091] ① Resuspend the bacteria (i.e., the precipitate obtained by low-speed centrifugation in step (1)) with 500 μL of reagent A (50 - 200 mg of wet bacteria are processed each time), centrifuge at 8000 g for 3 min, and discard the supernatant;

[0092] ② Resuspend the bacteria with 500 μL of extraction buffer B, ultrasonically disrupt it at 150 - 300 W for 5 - 15 min, and centrifuge at 10000 g for 10 min to collect the supernatant;

[0093] ③ Add 500 μL of C extraction solution to the supernatant obtained in step ②, vigorously rotate for 20 s to mix evenly, water bath at 66 - 68 °C for 30 min, and gently mix with a pipette every 10 min; finally, let it stand overnight at 4 °C, centrifuge at 3000 g for 20 min, and take the uppermost supernatant, which is the LPS solution. The LPS concentration measured by the sulfuric acid - phenol method is 150 μg / mL;

[0094] (3)Synthesize DMON

[0095] Under stirring conditions, first add 68 mg of TEA to 25 mL of water, gently stir in an 80 °C oil bath for 0.5 h to ensure the uniform dispersion and full dissolution of TEA; then add 380 mg of CTAB and 168 mg of NaSal to the above solution and continue to stir in an 80 °C oil bath for 1 h to fully dissolve CTAB and NaSal and form a stable template structure; then, mix 2 mL of TEOS and 1.6 mL of BTEE and add them to the above water - CTAB - NaSal - TEA, continue to stir in an 80 °C oil bath for 1 h. After the reaction is completed, the reaction product is reflux - extracted with HCl and methanol solution (V HCl : V 甲醇 = 1:9) at 60 °C for 6 h, and extract 3 times in total to remove the template agent. Finally, dry it overnight under vacuum at room temperature to obtain DMON powder;

[0096] (4)Prepare DMON - NH 2

[0097] Mix the DMON powder obtained in step (3) and absolute ethanol to obtain a DMON ethanol solution with a concentration of 10 mg / mL; take 5 mL of the DMON ethanol solution and mix it with 5 μL of APTES, stir and react at 500 rpm at room temperature for 12 h to ensure the full progress of the reaction; after the reaction is completed, centrifuge the reaction product at 10000 rpm, wash the precipitate with an appropriate amount of water to remove the unreacted APTES, and dry the washed reaction product under vacuum at room temperature for 12 h to obtain positively charged amino - modified DMON - NH 2 ;

[0098] (5)Prepare LPS@DMON@OMV

[0099] Mix the DMON - NH 2 obtained in step (4) and water to obtain a DMON - NH 2 solution with a concentration of 10 mg / mL; mix 500 μL of the LPS solution (150 μg / mL) obtained in step (3) and 2 mL of DMON - NH 2Mix with the solution (10 mg / mL), stir and react at 500 rpm at room temperature for 12 h to load LPS on DMON through electrostatic interaction; after the reaction is completed, centrifuge the reaction product at 10,000 rpm, wash it with an appropriate amount of water, and redissolve the washed reaction product with 2 mL of 1×PBS buffer solution to obtain the LPS@DMON solution; take 500 μL of the obtained LPS@DMON solution and mix it with 500 μL of the OMV solution (concentration of 1 mg / mL) at 4°C, and extrude it back and forth 10 times through a 200 nm polycarbonate membrane using an Avanti mini-liposome extruder, and finally centrifuge at 4°C and 8,000 rpm for 10 min to centrifuge and separate the unloaded OMV to obtain a nano-vaccine based on Helicobacter pylori outer membrane vesicles (LPS@DMON@OMV).

[0100] Comparative Example 1

[0101] (1)-(4) are the same as Example 1;

[0102] (5) Prepare LPS@DMON@OMV

[0103] The dosage of the LPS solution is 1000 μL, which is twice that of Example 1, and other operations are the same as Example 1.

[0104] Comparative Example 2

[0105] (1)-(4) are the same as Example 1;

[0106] (5) Prepare LPS@DMON@OMV

[0107] The dosage of the LPS solution is 167 μL, which is 1 / 3 of Example 1, and other operations are the same as Example 1.

[0108] Comparative Example 3

[0109] (1)-(4) are the same as Example 1;

[0110] (5) Prepare LPS@DMON@OMV

[0111] The dosage of the OMV solution (concentration of 2 mg / mL) is 500 μL, which is twice that of Example 1, and other operations are the same as Example 1.

[0112] Comparative Example 4

[0113] (1)-(4) are the same as Example 1;

[0114] (5) Prepare LPS@DMON@OMV

[0115] The dosage of the OMV solution (concentration of 0.5 mg / mL) is 500 μL, which is 1 / 2 of Example 1, and other operations are the same as Example 1.

[0116] Comparative Example 5

[0117] (1)-(4) are the same as in Example 1;

[0118] (5) Prepare LPS@DMON@OMV

[0119] DMON-NH 2 (with a concentration of 20 mg / mL) The dosage is 2 mL, which is twice that of Example 1, and other operations are the same as in Example 1.

[0120] Effect Example 1

[0121] The dosages of LPS, OMV and DMON-NH in Example 1 and Comparative Examples 1-5 2 The dosage (mass) ratio is shown in Table 1.

[0122] Table 1 Dosage ratios of LPS, OMV and DMON-NH in Example 1 and Comparative Examples 1-5 2 Dosage ratio

[0123] Control Group <![CDATA[LPS:OMV:DMON-NH 2 > Example 1 0.75:20:200 Control Example 1 1.5:20:200 Control Example 2 0.25:20:200 Control Example 3 0.75:40:200 Control Example 4 0.75:10:200 Control Example 5 0.75:20:400

[0124] Among them, compared with Example 1, the increase in the usage amount of LPS in Comparative Example 1 will lead to an increase in the toxicity of the final product, and the cell survival rate in the in vitro experiment of the prepared nano-vaccine is greatly reduced. Among them, when the experimental concentration of OMV is 20 μg / mL, the CCK-8 cell toxicity experiment (the specific detection method refers to Effect Example 2) shows that the cell survival rate of Comparative Example 1 is 64%, which is 33% lower than that of Example 1.

[0125] Compared with Example 1, the insufficient usage amount of LPS in Comparative Example 2 will lead to a lower level of immune cytokines in the in vitro experiment, that is, the immune effect decreases. Among them, when the experimental concentration of OMV is 20 μg / mL, the quantitative detection results of in vitro immune cytokines show (the specific detection method refers to Effect Example 2): the levels of IFN-γ, IL-4 and IL-17A in the LPS@DMON@OMV group of Comparative Example 2 have no significant difference compared with the DMON@OMV group, and are significantly lower than those of Example 1;

[0126] Compared with Example 1, the increase in the usage amount of OMV in Comparative Example 3 has a limited increase in the protein loading rate. Among them, compared with Example 1, the protein loading rate of OMV increases by 13%, that is, it will cause a waste of antigen cost and a significant increase in raw material cost;

[0127] Compared with Example 1, the insufficient OMV in Comparative Example 4 leads to an unsaturated vector, that is, a low protein loading rate. Compared with Example 1, the protein loading rate (the specific detection method refers to Effect Example 2) is reduced by 60%, and the utilization rate is low, affecting the immune effect;

[0128] Compared with Example 1, an excessive amount of DMON-NH in Comparative Example 5 2 will cause the extruder to clog, resulting in a decrease in the extrusion passing rate.

[0129] In summary, for the mass ratio of LPS, OMV and DMON-NH 2 being 0.75:20:200, a relatively stable protein and LPS loading ratio can be obtained.

[0130] Comparative Example 6

[0131] (1) Prepare an OMV solution with reference to Example 1;

[0132] (2) Prepare DMON powder with reference to Example 1, mix the DMON powder and water to obtain a DMON aqueous solution with a concentration of 10 mg / mL;

[0133] (3) Mix 500 μL of the DMON aqueous solution (10 mg / mL) with 500 μL of the OMV solution (1 mg / mL) at 4°C, and use an Avanti mini-liposome extruder to extrude back and forth through a 200 nm polycarbonate membrane 10 times. Finally, centrifuge at 4°C and 8000 rpm for 10 min to centrifuge and separate the unloaded OMV to obtain DMON@OMV.

[0134] Effect Example 2

[0135] I. Test method

[0136] Using the OMV, LPS, DMON, DMON-NH 2 prepared in Example 1, LPS@DMON and LPS@DMON@OMV, as well as DMON@OMV prepared in Comparative Example 6 as objects, conduct the following tests:

[0137] 1.1 Sample test characterization

[0138] 1.1.1 Morphology and Zeta potential

[0139] (1) Observe the morphologies of OMV, DMON and LPS@DMON@OMV prepared in Example 1 by transmission electron microscopy (TEM). Place the samples on 200-mesh copper grids, stain them with 2% phosphotungstic acid negative staining solution (Regen Biotech, product number: DZ0035) respectively, and then obtain TEM images at 80 kv.

[0140] (2) Use dynamic light scattering (DLS) to measure OMV, LPS, DMON, DMON-NH 2Surface potential of LPS@DMON, DMON@OMV, and LPS@DMON@OMV.

[0141] 1.1.2 Membrane protein assessment

[0142] (1) The total protein content of OMV on the nanoparticles was determined using the Bradford protein Assay Kit (BCA) and compared with the standard BCA protein to calculate the protein content.

[0143] (2) OMV, DMON, DMON@OMV, and LPS@DMON@OMV were subjected to SDS-PAGE and Coomassie brilliant blue staining according to the conventional method.

[0144] 1.1.3 LPS concentration determination

[0145] The amount of LPS used was determined by the sulfuric acid-phenol method (Wang Xitong, Yang Jiaxiang, Wang Aijia, et al. Rapid determination of the polysaccharide content in the rind of watermelon by the micro phenol-sulfuric acid method [J]. Journal of Food Safety and Quality, 2024, 15(22): 204-210. DOI: 10.19812 / j.cnki.jfsq11-5956 / ts.20240917002.). The principle is as follows: Under the action of concentrated sulfuric acid, polysaccharides are hydrolyzed into monosaccharides, and monosaccharides are rapidly dehydrated into formaldehyde derivatives; then the formaldehyde derivatives condense with phenol to form a stable orange-yellow compound. In a certain concentration range, the absorbance at 490 nm has a linear relationship with the polysaccharide content. A standard curve was plotted with glucose concentration (X) and absorbance (Y) to calculate the LPS content per unit mass of LPS@DMON@OMV.

[0146] 1.2. In vitro cell tests

[0147] 1.2.1 Cytotoxicity detection

[0148] Mouse macrophages RAW264.7 (Cell Bank of the Chinese Academy of Sciences (Shanghai, China)) in the logarithmic growth phase were seeded in 96-well plates (100 μL / well) at a density of 5×10 3 cells / well. After the cells were completely adherent, DMEM complete medium containing LPS@DMON@OMV was added respectively (final concentration gradient of OMV: 0, 10, 20, 40, 60, 80 μg / mL), and cell-free medium was used as the blank control; after culturing the samples in each group at 37 °C and 5% CO 2 for 24 h, 10 μL of CCK-8 working solution was added to each well, and the incubation was continued for 1 h under light-proof conditions; the absorbance of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument to calculate the relative cell viability.

[0149] 1.2.2 Analysis of cell phagocytosis effect

[0150] According to the instruction manual, the OMV and LPS@DMON@OMV nanoparticles were labeled with the exosome fluorescent dye DIR (Umibio, catalog number UR21017). Mouse macrophages RAW264.7 in good growth state were seeded in 6-well plates at a density of 5×10 5 cells / well. After the cells adhered, the above-mentioned DIR-fluorescently labeled OMV and LPS@DMON@OMV nanoparticles (final concentration of OMV 20 μg / mL) were added respectively, and cells with PBS added were used as the control group. After co-culturing for 6 h, the culture supernatant was removed, and the cells were washed 3 times with PBS to remove uninternalized nanoparticles. An appropriate amount of 4% paraformaldehyde fixative (Biosharp, catalog number BL539A-1) was added to fix the cells for 20 min, and then the cells were washed 2 times with PBS. Subsequently, DAPI staining solution (working concentration 1 μg / mL) was used for nuclear staining for 15 min. After washing with PBS to remove the residual dye, multi-channel fluorescence imaging was performed using a laser confocal scanning microscope (CLSM). The DIR-labeled nanoparticles showed red fluorescence (excitation / emission wavelength: 748 / 780 nm), and DAPI nuclear staining showed blue fluorescence (excitation / emission wavelength: 358 / 461 nm).

[0151] 1.2.3 Quantitative detection of immunocytokines

[0152] Mouse macrophages RAW264.7 were seeded in 96-well plates at a density of 5×10 3 cells / well. The experimental groups were treated with OMV, DMON@OMV, and LPS@DMON@OMV respectively (n = 6). The final concentration of the antigen OMV in each group was 20 μg / mL, and cells with PBS added were used as the control group. After culturing for 24 h, the cell debris was removed by centrifugation at 1200×g for 10 min at 4°C, and the supernatant was taken. According to the operation instructions of the ELISA kit, the secretion levels of IFN-γ, IL-4, and IL-17A were quantitatively detected. Among them, the mouse interferon-γ (IFN-γ) kit (catalog number RX203097M), mouse interleukin-4 (IL-4) kit (catalog number RX203051M), and mouse interleukin-17A (IL-17A) kit (catalog number RX203066M) were all purchased from Ruixin Biology. The standard curve was drawn and the concentration was calculated strictly in accordance with the technical specifications of the kit.

[0153] 1.3 Animal immune experiment evaluation

[0154] 1.3.1 Immunization procedure

[0155] Six-week-old female C57BL / 6 mice (weighing 16 - 22 g, purchased from Guangdong Provincial Medical Laboratory Animal Center) were adaptively fed in an SPF-level environment for 14 days and then randomly divided into 4 groups (n = 4 / group), named as: ① PBS; ② OMV; ③ DMON@OMV; ④ LPS@DMON@OMV; Except for the PBS group, each group was orally gavaged with the corresponding antigen preparation solution on days 1, 7, and 14. Among them, the volume of each gavage of the antigen preparation solution was 100 μL, and the dosage of the antigen preparation was 40 μg (calculated based on the amount of OMV protein). Samples were collected and analyzed 7 days after the last immunization. The specific methods are as follows:

[0156] (1)Serum samples: Whole blood was collected from the orbital venous plexus, left to stand at 37 °C for 1 h, then left overnight at 4 °C, centrifuged at 3000 g for 15 min, the supernatant serum was aliquoted and stored at -80 °C for later use;

[0157] (2)Vaginal secretion samples: The vaginal cavity was repeatedly rinsed with 100 μL of PBS, the collected fluid was centrifuged at 4000 g for 10 min, and the supernatant was taken and stored at -80 °C for later use;

[0158] (3)Gastric tissue homogenate: The mice were sacrificed, and the gastric tissue of the mice was taken out under sterile conditions, rinsed with pre-cooled PBS to remove residual blood, weighed, and then the gastric tissue was minced; The gastric tissue was homogenized in PBS containing 0.5 mM PMSF protease inhibitor (Beyotime, product number ST506) at a weight-to-volume ratio of 1:9, ground thoroughly on ice, and finally the homogenate was centrifuged at 5000×g for 10 min, and the supernatant was collected and stored at -80 °C for later use;

[0159] (4)Spleen cell isolation: The mice were sacrificed, and the spleens of the mice were taken out under sterile conditions and placed in a 6-cm cell culture dish containing 5 mL of 1640 medium; Cells were separated by mechanical dissociation method. The plunger of a syringe was used to push the spleen tissue through a 40-μm cell sieve, and then the cell suspension was used to thoroughly rinse the sieve. The cells were centrifuged at 600×g for 5 min at 4 °C, and the supernatant was discarded; The cells were resuspended with 1 mL of 1× red blood cell lysis buffer (Beyotime, product number C3702), incubated at room temperature for 5 min, and gently shaken or rotated occasionally during this period; 3 mL of 1640 medium was added to terminate the lysis reaction, centrifuged at 600×g for 5 min, the supernatant was discarded, and the cells were resuspended with PBS and adjusted to a cell density of 1×10 7 cells / mL;

[0160] (5)The main organs (heart, liver, spleen, lung, kidney) and blood of each group were collected, with 4 mice in each group.

[0161] 1.3.2 Analysis of anti-Helicobacter pylori IgG titer

[0162] (1)Coated antigen: Dilute Helicobacter pylori SS1 to 10 8 CFU / mL with carbonate buffer (pH 9.6), add 100 μL to each well of a 96-well plate, and coat overnight at 4°C;

[0163] (2)Blocking: Discard the coating solution, wash the plate 3 times with PBST, add 300 μL of PBS solution containing 3% (mass percentage) skim milk powder to each well, and block at 37°C for 1 h;

[0164] (3)Incubation of serum samples: Take out the plate, flick out the blocking solution, wash the plate 5 times, serially dilute the serum samples (prepared in step 1.3.1(1)) with PBS (100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600-fold), 100 μL / well, use PBS as a negative control, and incubate at 37°C for 1 h;

[0165] (4)Incubation of enzyme-labeled secondary antibody: Take out the plate, flick out the liquid in the plate, wash the plate 5 times with PBST, add the secondary antibody of HRP-labeled goat anti-mouse diluted 8000-fold with PBS (Goat Anti-Mouse HRP-conjugated Goat Anti-Mouse IgG(H+L), Proteintech, catalog number: SA00001-1), 100 μL / well, and incubate at 37°C for 1 h;

[0166] (5)Color development and termination: Flick out the enzyme-labeled secondary antibody in the plate, wash the plate 5 times with PBST, pat dry with absorbent paper, add 100 μL / well of TMB color development solution (Solarbio, catalog number: PR1200), develop color in the dark at 37°C for 15 min, immediately add 50 μL / well of 2M sulfuric acid termination solution, and read the OD value at 450 nm using an enzyme-linked immunosorbent assay reader;

[0167] (6)Titer calculation: Judgment threshold = mean OD value of negative control × 2.1, the sample titer is defined as the reciprocal of the highest dilution factor that produces a threshold signal. Plot the antibody titer curve with the dilution factor on the x-axis and the OD value on the y-axis.

[0168] 1.3.3 Evaluation of the specificity of anti-Helicobacter pylori antibodies

[0169] Specific analysis of anti-Helicobacter pylori antibodies in the serum of the LPS@DMON@OMV group prepared in step 1.3.1(1) was performed using three kinds of bacilli, namely Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, and Acinetobacter baumannii ATCC 19606. PBS was set as the negative control, and the specific steps were referred to step 1.3.2. An antibody titer curve was plotted with the dilution factor as the abscissa and the OD value as the ordinate.

[0170] 1.3.4 Determination of mucosal immune IgA level

[0171] The IgA levels in the vaginal secretion supernatant prepared in step 1.3.1(2) and the gastric homogenate supernatant prepared in step 1.3.1(3) were quantitatively detected according to the operation instructions of the ELISA kit (Mouse Immunoglobulin A (IgA) Kit, Ruixin Bio, catalog number RX202739M). The standard curve drawing and concentration calculation were strictly in accordance with the technical specifications of the kit.

[0172] 1.3.5 Analysis of splenocyte immune factors

[0173] The splenocytes prepared in step 1.3.1(4) were seeded in a 24-well plate at a density of 1×10 7 cells / well, 500 μL / well, and OMV, DMON@OMV, and LPS@DMON@OMV were added to enhance the stimulation of mouse splenocytes. Among them, the final concentration of the antigen OMV was 10 μg / mL, and the wells without antigen were used as the control. After culturing for 24 h, the cell debris was removed by centrifugation at 1200×g for 10 min at 4°C, and the supernatant was taken. The levels of IFN-γ, IL-4, and IL-17A in the cell supernatant were quantitatively detected according to the operation instructions of the ELISA kit (same as 1.2.3). The standard curve drawing and concentration calculation were strictly in accordance with the technical specifications of the kit.

[0174] 1.3.6 Safety evaluation

[0175] Taking the main organs (heart, liver, spleen, lung, kidney) of each group collected 7 days after the last immunization as the object, the tissues were embedded in paraffin and sectioned for HE staining; the blood collected 7 days after the last immunization was subjected to routine blood tests.

[0176] II. Test results

[0177] 2.1 Results of sample test characterization

[0178] 2.1.1 TEM and Zeta potential results

[0179] Figure 1 TEM results of OMV, DMON, and LPS@DMON@OMV. As can be seen from Figure 1 it, a film has formed on the surface of LPS@DMON@OMV particles, with a diameter of approximately 150 - 200 nm.

[0180] Figure 2 are the surface Zeta potentials of each component and intermediate during the synthesis of the nano - vaccine to verify the stable generation of the vaccine. As can be seen from Figure 2 it, after loading LPS, the value of DMON - NH 2 (surface - modified with amino groups) changes from 24.1 ± 0.5 mV to - 13.6 ± 0.4 mV (LPS@DMON), which is mainly the result of charge absorption. The Zeta potential indicates that the coating of OMV is successful because after the coating is completed, due to the electronegativity of OMV, the electronegativity of LPS@DMON@OMV increases to - 27.9 ± 0.9 mV.

[0181] 2.1.2 Protein analysis and LPS concentration determination

[0182] Figure 3 are the SDS - PAGE result graphs of the protein components of OMV, DMON, DMON@OMV, and LPS@DMON@OMV. The membrane coating is further verified by gel electrophoresis. The results show that the membrane protein composition of Helicobacter pylori outer membrane vesicles is retained throughout the preparation process. No protein is detected in DMON nanoparticles, and the protein composition content of LPS@DMON@OMV has no significant difference from that of DMON@OMV, indicating that the loading of LPS on OMV has no effect.

[0183] In addition, the total protein content of OMV on LPS@DMON@OMV nanoparticles is measured using the Bradford protein Assay Kit (BCA). The results show that the OMV protein loading ratio (the weight ratio of the immobilized protein on the nanoparticles) is approximately 38.2 ± 0.5 μg / mg; the loading amount of LPS in LPS@DMON@OMV nanoparticles is measured by the sulfuric acid - phenol method to be approximately 24.8 ± 3.7 μg / mg.

[0184] 2.2 In vitro cell experiment results

[0185] 2.2.1 Cytotoxicity detection

[0186] The proliferative effect of LPS@DMON@OMV nanoparticles on macrophages is studied by CCK - 8, and the results are shown in Figure 4 . As can be seen from the figure, LPS@DMON@OMV nanoparticles have no significant cytotoxicity to macrophages when the concentration is below 40 μg / mL.

[0187] 2.2.2 Cell uptake results

[0188] OMV and LPS@DMON@OMV were stained with DIR dye, and the cell nuclei were stained with DAPI. The uptake of mouse macrophages RAW264.7 was observed by laser confocal microscopy. The results are shown in Figure 5 . It can be seen from the figure that the cells successfully took up OMV and LPS@DMON@OMV, and the LPS@DMON@OMV group showed a stronger red fluorescence signal. This indicates that, compared with OMV, the LPS@DMON@OMV nanoparticles can promote the uptake of the vaccine by macrophages.

[0189] 2.2.3 Effects on the secretion of factors by mouse macrophages RAW264.7

[0190] ELISA was used to detect the levels of IFN-γ, IL-4 and IL-17A secreted by mouse macrophages RAW264.7 after different drug treatments (OMV, DMON@OMV and LPS@DMON@OMV). The results are shown in Figure 6 . It can be seen from the figure that, compared with the OMV and DMON@OMV groups, the LPS@DMON@OMV group significantly increased the levels of the cytokines IFN-γ, IL-4 and IL-17A. Therefore, LPS@DMON@OMV can significantly promote the secretion of immune cytokines by mouse macrophages RAW264.7.

[0191] 2.3 Animal immune effect study

[0192] 2.3.1 Serum anti-Helicobacter pylori IgG titer

[0193] As Figure 7 shown, the ELISA results showed that the titer of anti-Helicobacter pylori specific antibodies induced by the LPS@DMON@OMV group was significantly higher than that of the three control groups. Among them, the antibody titer of the OMV group was 1:800; the antibody titer of the DMON@OMV group was 1:1600; the antibody titer of the LPS@DMON@OMV group was 1:12800. This indicates that the vaccine LPS@DMON@OMV can stimulate stronger humoral immunity.

[0194] 2.3.2 Specificity of anti-Helicobacter pylori antibodies

[0195] As Figure 8 shown, the anti-Helicobacter pylori antibody titer was significantly higher than that of Escherichia coli, Salmonella typhimurium and Acinetobacter baumannii, indicating that the anti-Helicobacter pylori antibodies induced by the LPS@DMON@OMV group had high specificity.

[0196] 2.3.3 Mucosal immune IgA level

[0197] Mucosal immunity is the first line of defense of the host immune system, Figure 9 which is the IgA level in the supernatant of vaginal secretions and gastric homogenates after immunizing mice. It can be seen from the figure that the IgA antibody levels in the vaginal secretions and gastric mucosa of all experimental group mice are significantly different from those of the PBS control group. The IgA levels in the supernatant of vaginal secretions and gastric homogenates of the LPS@DMON@OMV group are significantly higher than those of the other 3 control groups, indicating that this vaccine can stimulate stronger mucosal immunity.

[0198] 2.3.4 Levels of immune factors in splenocytes

[0199] IFN-γ reflects Th1-type immune response, IL-4 reflects Th2-type immune response, and IL-17A is an index of Th17-type immune response. Figure 10 This is the result analysis chart of immune factors IFN-γ, IL-4, and IL-17A in splenocytes of immunized mice. It can be seen from the figure that the secretion levels of cytokines IFN-γ, IL-4, and IL-17A after immunization are all significantly higher than those of the PBS control group, and the secretion level of the LPS@DMON@OMV group is significantly higher than that of the other 3 control groups, indicating that this vaccine can produce an effective mixed Th1 / Th2 / Th17 immune response.

[0200] 2.3.5 Effects of immunization on blood immune cells

[0201] The results of blood routine analysis ( Figure 11 ) show that the levels of white blood cells (WBC), lymphocytes (LYM), neutrophils (NEU), and monocytes (MON) in mice after LPS@DMON@OMV immunization are all significantly increased. There is no significant difference in the single OMV group and the DMON@OMV group, indicating that the cellular immune level of animals after LPS@DMON@OMV immunization is significantly enhanced.

[0202] 2.3.6 Safety evaluation

[0203] Figure 12 These are the results of red blood cell content (RBC), hemoglobin content (HGB), and platelet content (PLT) in blood routine analysis. The results show that there is no significant change in these index levels.

[0204] In addition, the hearts, livers, spleens, lungs, and kidneys of mice were subjected to HE staining, and the results are shown in Figure 13 . It can be seen from the figure that compared with the PBS control group, there are no lesions in the visceral tissues of immunized mice, and there is no significant difference in each organ. The results support the safety of the LPS@DMON@OMV nano-vaccine.

[0205] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles, characterized in that The following steps are involved: (1) Isolation and purification of OMV Take the Helicobacter pylori bacterial suspension, centrifuge at 8000-10000rpm, obtain the supernatant and precipitate respectively; filter the supernatant and take the filtrate; ultracentrifuge the filtrate at 100000-300000g, discard the supernatant, resuspend the precipitate, and obtain the OMV solution; (2) Extraction of LPS Take the precipitate obtained by low-speed centrifugation in step (1), extract LPS according to conventional methods, and obtain LPS solution; (3) Synthetic DMON DMON was prepared by a one-pot method using cationic surfactants CTAB and NaSal as templates, TEOS and BTEE as silica sources, and TEA as a catalyst. (4) Preparation of DMON-NH2 The DMON obtained in step (3) is mixed with ethanol to obtain a DMON ethanol solution; the DMON ethanol solution is mixed with APTES to carry out an amination reaction, and after the reaction is completed, the reaction product is purified and dried to obtain DMON-NH2; (5) Preparation of LPS@DMON@OMV The DMON-NH2 prepared in step (4) is mixed with water to obtain a DMON-NH2 solution; the LPS solution prepared in step (2) and the DMON-NH2 solution are mixed and stirred for reaction; after the reaction is completed, the reaction product is purified and resuspended with a buffer solution to obtain an LPS@DMON solution; the LPS@DMON solution and the OMV solution prepared in step (1) are mixed and then extruded 10-20 times using a liposome extruder, and finally centrifuged to obtain a nanovaccine based on Helicobacter pylori outer membrane vesicles; The mass ratio of LPS in the LPS solution, OMV in the OMV solution, and DMON-NH2 in the DMON-NH2 solution described in step (5) is 0.75:(20-40):

200.

2. The method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 1, characterized in that: The conditions of the low-speed centrifugation in step (1) are: 4°C, 8000-10000 rpm, 2-3 times, each time for 10-20 min; The ultracentrifugation conditions in step (1) are: 4°C, 100,000-300,000 g for 1.5-2.5 h.

3. The method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 1, characterized in that: The method for preparing DMON described in step (3) specifically comprises the following steps: ① Under stirring conditions, add TEA to water and stir in an oil bath at 70-90℃ for 0.3-0.7h; add CTAB and NaSal to the solution after stirring in the oil bath, and continue stirring in the oil bath at 70-90℃ for 0.8-1.2h to fully dissolve CTAB and NaSal and form a stable template structure; ② After mixing TEOS and BTEE, add them to the system in step (1), and continue stirring in a 70-90° C. oil bath for 0.5-1.5 h; after the reaction is completed, purify and dry the reaction product to obtain DMON.

4. The method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 3, characterized in that: The mass ratio of TEA, CTAB and NaSal described in step ① is (60-80): (350-400): (150-180); The volume ratio of TEOS and BTEE in step ② is (1.5-2.5): (1.4-1.8); The mass volume ratio of TEA described in step ① and TEOS described in step ② is (60-80) mg:mL:(1.5-2.5).

5. The method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 1, characterized in that: The volume ratio of the LPS solution to the DMON-NH2 solution in step (5) is (1:4)-(1:5); The mass ratio of LPS in the LPS solution to DMON-NH2 in the DMON-NH2 solution in step (5) is 0.075:20; The concentration of the LPS solution described in step (5) is 150 μg / mL.

6. The method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 1, characterized in that: The volume ratio of the LPS@DMON solution to the OMV solution in step (5) is 1:1; The mass ratio of DMON-NH2 in the LPS@DMON solution to OMV in the OMV solution described in step (5) is 5:0.5; The concentration of the OMV solution described in step (5) is 1 mg / mL.

7. The method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 1, characterized in that: The reaction conditions in step (5) are stirring at 300-600 rpm for 12-24 hours.

8. A nano vaccine based on Helicobacter pylori outer membrane vesicles, characterized in that It is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the nano vaccine based on Helicobacter pylori outer membrane vesicles according to claim 8 in the preparation of products for preventing and treating peptic ulcer, chronic gastritis and gastric cancer.

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