Nano vaccine based on helicobacter pylori outer membrane vesicles as well as preparation method and application of nano vaccine

Abstract

The invention belongs to the field of vaccine preparation, and particularly relates to a nano vaccine based on helicobacter pylori outer membrane vesicles as well as a preparation method and application of the nano vaccine. OMV and LPS of helicobacter pylori are used as antigens and adjuvants, DMON is used as a carrier, then electrostatic interaction is utilized to enable the DMON to adsorb LPS micromolecules, then a liposome extruder is utilized to uniformly wrap the helicobacter pylori OMV on the surface of a DMON nano material, and the nano vaccine based on helicobacter pylori outer membrane vesicles is obtained. In-vivo and in-vitro tests prove that the nano vaccine can be ingested by macrophages, the phagocytic ability of the macrophages is promoted, the level of secreting various immune cell factors by the macrophages is remarkably improved, and high-level antigen-specific humoral immunity, mucosal immunity and Th1 / Th2 / Th17 type cellular immune response can be induced in a mouse body.

Description

Technical Field

[0001] 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. Background Art

[0002] Helicobacter pylori is a unipolar, multi-flagellated, spirally curved, Gram-negative, microaerophilic bacterium that can move in the gastric mucus layer and secrete urease to decompose urea to produce ammonia, adapting to the acidic environment of the stomach. Most Helicobacter pylori infections are usually asymptomatic, but this condition can persist as a chronic infection and increase the risk of peptic ulcers, chronic gastritis, and gastric cancer. Currently, antibiotic treatment is the most commonly used regimen for the treatment of Helicobacter pylori infection. In clinical practice, triple or quadruple therapy is generally used. However, with the popularization of antibiotic treatment, antibiotic resistance has become another prominent clinical problem. The development of safe and effective vaccines has become an important measure to prevent Helicobacter pylori infection.

[0003] Outer membrane vesicles (OMVs) are spherical nanostructures with a diameter of 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 the body's innate and adaptive immune responses to bacterial infections, making them potential new vaccine candidates. OMVs cannot replicate themselves, solving the main safety issues in the application of traditional inactivated whole bacterial vaccines, and their nanosize is conducive to uptake by antigen-presenting cells.

[0004] There is now an increasing focus on the use of nanomaterials to treat drug-resistant bacterial infections. Compared with pure antigen vaccines, the advantages of nanomaterial vaccines mainly include: (1) nanocarriers can avoid 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 nanoscale enhances the accumulation of antigens in lymph nodes, further enhancing their immune response ability. Summary of the invention

[0005] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a method for preparing a nanovaccine based on Helicobacter pylori outer membrane vesicles, which method uses Helicobacter pylori outer membrane vesicles (OMV) as antigens, lipopolysaccharide (LPS) as adjuvant, dendritic mesoporous organic silica (DMON) as a carrier, and utilizes electrostatic interaction to make DMON adsorb LPS small molecules; finally, a liposome extruder is used to evenly wrap OMV on the surface of DMON nanomaterial to obtain a nanovaccine based on Helicobacter pylori outer membrane vesicles.

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

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

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for preparing a nano vaccine based on Helicobacter pylori outer membrane vesicles comprises the following steps:

[0010] (1) Isolation and purification of OMV

[0011] 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;

[0012] (2) Extraction of LPS

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

[0014] (3) Synthetic DMON

[0015] DMON was prepared by a one-pot method using cationic surfactants hexadecyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) as templates, tetraethyl orthosilicate (TEOS) and 1,2-bis(triethoxysilyl)ethane (BTEE) as silica sources, and triethanolamine (TEA) as a catalyst.

[0016] (4) Preparation of DMON-NH2

[0017] The DMON obtained in step (3) is mixed with ethanol to obtain a DMON ethanol solution; the DMON ethanol solution is mixed with 3-aminopropyltriethoxysilane (APTES) to carry out an amination reaction, and after the reaction is completed, the reaction product is purified and dried to obtain DMON-NH2;

[0018] (5) Preparation of LPS@DMON@OMV

[0019] 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 remove unloaded OMVs to obtain a nanovaccine 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 and culture it at 37°C in a microaerobic environment for 48-72 hours, then collect the colonies and add them to liquid culture medium for expansion culture, and continue to culture for 48-72 hours;

[0022] The specific conditions of the micro-oxygen environment are preferably 5% O2, 10% CO2, and 85% N2;

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

[0024] The conditions of the low-speed centrifugation in step (1) are preferably: 4°C, 8000-10000 rpm low-speed centrifugation 2-3 times, each time for 10-20 min, taking the supernatant after each centrifugation, and centrifuging 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 LPS extraction;

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

[0026] The ultracentrifugation conditions in step (1) are preferably 4°C, 100,000-300,000 g ultracentrifugation for 1.5-2.5 h;

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

[0028] The method for preparing DMON described in step (3) specifically comprises the following steps:

[0029] 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 above solution and continue stirring in an oil bath at 70-90℃ for 0.8-1.2h to fully dissolve CTAB and NaSal and form a stable template structure;

[0030] ② Mix TEOS and BTEE and add them to the system in step (1), and continue stirring in a 70-90°C oil bath for 0.5-1.5h; 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 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 purification described in step ② is preferably:

[0035] The reaction product obtained in step ② is subjected to reflux extraction using a mixed solution of hydrochloric acid and methanol; the volume ratio of the hydrochloric acid to methanol is (1:8)-(1:10); the conditions of the reflux extraction are preferably: a temperature of 55-65°C, repeated extraction 2-4 times, each extraction time 5-7 hours, to ensure that the template (structure directing agent) and the like are completely removed;

[0036] The drying condition described in step ② is preferably vacuum drying for 12-24 hours;

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

[0038] The conditions of the amination reaction in step (4) are preferably stirring at 300-600 rpm for 12-24 hours to ensure that the reaction proceeds fully;

[0039] The specific operation of purification and drying in step (4) is preferably:

[0040] After the reaction is completed, the solid and liquid are separated and washed with ethanol or water to remove unreacted APTES. The washed product is vacuum dried for 12-24 hours to obtain DMON-NH2;

[0041] The mass ratio of LPS in the LPS solution, OMV in the OMV solution, and DMON-NH2 in the DMON-NH2 solution in step (5) is preferably 0.75:(20-40):200;

[0042] The volume ratio of the LPS solution to the DMON-NH2 solution in step (5) is preferably (1:4)-(1:5);

[0043] The mass ratio of LPS in the LPS solution to DMON-NH2 in the DMON-NH2 solution in step (5) is preferably 0.075:20;

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

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

[0046] The mass ratio of DMON-NH2 in the LPS@DMON solution to OMV in the OMV solution in step (5) is preferably 5:0.5;

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

[0048] The reaction conditions in step (5) are preferably stirred at 300-600 rpm for 12-24 hours;

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

[0050] After the reaction is completed, the solid and liquid are separated and washed with water to remove unreacted LPS, and the washed product is resuspended in PBS buffer solution;

[0051] The mixing and extrusion of the LPS@DMON solution and the OMV solution in step (5) is preferably carried out at 4°C;

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

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

[0054] The use of the 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, wherein the vaccine uses Helicobacter pylori OMV as an antigen, LPS as an adjuvant, and DMON as a carrier; wherein LPS as an adjuvant can improve the immunogenicity of OMV and enhance the immune effect; DMON has multi-level nanopores and a dendritic structure, so that it has a larger specific surface area for displaying antigens and adsorbing adjuvant small molecules.

[0057] Since LPS and DMON are negatively charged, the present invention first uses APTES (3-aminopropyltriethoxysilane) to perform amino modification on DMON to make it positively charged; then DMON-NH2 is loaded with LPS, which is a small molecule structure, through electrostatic interaction, so that it is adsorbed to the pores and surface of DMON to form LPS@DMON; then the antigen OMV and LPS@DMON are further mixed and extruded, and the antigen OMV will wrap LPS@DMON to form a stable round nanoparticle vaccine; if OMV and DMON are mixed first, OMV will wrap DMON to form a film on its surface, then LPS cannot be loaded onto the entire nanoparticle, but is dispersed in the solution, and then centrifuged, and the final product does not contain LPS; in addition, the present invention chooses OMV to wrap LPS@DMON because OMV has certain natural biocompatibility and targeting, and is easily actively taken up by antigen presenting cells, so other components are wrapped in OMV.

[0058] In addition, the dosage relationship of LPS, OMV and DMON is very important. The ratio provided by the present invention can obtain a relatively stable protein and LPS loading ratio. Too much LPS will increase toxicity, too little will not be effective; too much OMV will be wasteful, too little will have a low protein concentration; too much DMON will not pass the extruder easily. The appropriate dosage ratio can improve safety and avoid waste of raw materials.

[0059] Compared with the prior art, the present invention has the following advantages and effects:

[0060] (1) The synergistic effect of multiple components breaks through the limitation of insufficient immunogenicity of a single antigen. The present invention uses the natural OMV of Helicobacter pylori as the core antigen, retains the multiple antigenic epitopes of the pathogen (such as outer membrane proteins, lipoproteins, etc.), and can induce more comprehensive humoral immunity and cellular immunity compared to traditional single recombinant protein antigens. Animal experiments have verified that 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 utilizes Helicobacter pylori homologous lipopolysaccharide (LPS) as a natural adjuvant, avoiding the toxicity risk 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 Transmission electron micrographs of OMV, DMON and LPS@DMON@OMV, where A: OMV, B: DMON, C: LPS@DMON@OMV.

[0064] Figure 2 This is the surface Zeta potential analysis diagram of each component.

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

[0066] Figure 4 This is the analysis result of LPS@DMON@OMV toxicity test on macrophages.

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

[0068] Figure 6 This is the analysis result of immune cytokine secretion after macrophages were treated with OMV, DMON@OMV and LPS@DMON@OMV respectively.

[0069] Figure 7 This is an analysis graph of the anti-Helicobacter pylori antibody titer curve in the serum of mice after immunization.

[0070] Figure 8 This is an analysis chart of the specific detection results of anti-Helicobacter pylori antibodies in the serum of mice after immunization.

[0071] Fig. 9 It is the result analysis diagram of IgA level of vaginal secretion supernatant and gastric homogenate supernatant, wherein A: vaginal secretion supernatant, B: gastric homogenate supernatant.

[0072] Fig.10 This is the analysis chart of the results of spleen cell immune factor analysis.

[0073] Fig.11 This is an analysis chart of the results of routine blood analysis of the effects of immunization on each group of blood immune cells (white blood cells WBC, lymphocytes LYM, neutrophils NEU and monocytes MON).

[0074] Fig.12This is an analysis chart of the results of routine blood analysis of the effects of immunization on the red blood cell content (RBC), hemoglobin content (HGB) and platelet content (PLT) of each group.

[0075] Fig.13 These are the HE staining results of the heart, liver, spleen, lung, and kidney of each group of mice collected under a microscope. DETAILED DESCRIPTION

[0076] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments 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, et al. Effect of Helicobacter pylori infection on m6A levels in gastric cancer cells and its mechanism [J]. Journal of Shandong University (Medical Edition), 2023, 61(09): 10-18.);

[0078] Preparation of Columbia blood agar medium in the examples: Columbia agar medium (purchased from Guangdong Huankai Biotechnology Co., Ltd.) was sterilized according to the instructions for use, and 10% sterile defibrinated sheep blood was added to obtain Columbia blood agar medium.

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

[0080] Preparation of the Brucella broth medium in the examples: purchased Brucella broth dry powder medium (Guangdong Huankai Microbiological Technology Co., Ltd.) was prepared according to the instructions.

[0081] DMEM high glucose medium was purchased from Gibco (Cat. No.: C11995500BT).

[0082] DMEM complete culture medium: 90% DMEM + 10% fetal bovine serum (Gibco, catalog number C0235), plus 1% penicillin-streptomycin (Gibco, catalog number 15140122).

[0083] Example 1 Preparation of Helicobacter pylori Outer Membrane Vesicle Nanovaccine LPS@DMON@OMV

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

[0085] ① Liquid culture of Helicobacter pylori

[0086] The frozen Helicobacter pylori SS1 strain was taken out of the -80℃ freezer, thawed on ice, and 100μL of bacterial liquid was pipetted onto the freshly prepared Columbia blood agar medium, and evenly spread with a sterile spreading stick, and then inverted and cultured at 37℃ in a microaerobic environment (5% O2, 10% CO2, 85% N2) for 48h; then the colonies on the solid plate were collected with sterile 1×PBS buffer, and 500μL of OD 600nm =1.5 was placed in a 10 mL bacterial culture dish supplemented with 10% by volume of exosome-free fetal bovine serum from Brucella broth and cultured for 72 h;

[0087] ② Isolation and purification of OMV

[0088] Collection step ① Liquid culture of Helicobacter pylori bacterial suspension (OD 600nm =1-2), centrifuged at 4°C and 10000rpm for 20 minutes each time, and the supernatant was taken after each centrifugation, and the supernatant was centrifuged again; the supernatant obtained by the last centrifugation was collected, and the precipitates after each centrifugation were combined for subsequent LPS extraction; the supernatant obtained by the last centrifugation was filtered twice with a 0.22μm microporous filter, and the filtrate was taken; the filtrate was ultracentrifuged at 4°C and 200000g for 2h, the supernatant was discarded, and the precipitate was resuspended with sterile 1×PBS buffer and frozen at -80°C;

[0089] (2) Extraction of LPS

[0090] Take the precipitate obtained by low-speed centrifugation in step (1) and use a bacterial lipopolysaccharide extraction kit (Bebo Biotech, catalog number: BB-31302) to extract LPS (the LPS here is mainly LPS on Helicobacter pylori and LPS in some OMVs). The specific method is as follows:

[0091] ① Add 500 μL of reagent A (50-200 mg of wet bacteria per treatment) to resuspend the bacteria (i.e., the precipitate from low-speed centrifugation in step (1)), centrifuge at 8000 g for 3 min, and discard the supernatant;

[0092] ② Resuspend the bacteria with 500 μL of extract solution B, ultrasonically disrupt them at 150-300 W for 5-15 min, and centrifuge at 10,000 g for 10 min to collect the supernatant;

[0093] ③ Add 500 μL of C extract to the supernatant prepared in step ②, rotate vigorously for 20 seconds to mix evenly, place in a 66-68°C water bath for 30 minutes, and gently mix with a pipette every 10 minutes to mix evenly; finally, after standing at 4°C overnight, centrifuge at 3000g for 20 minutes, take the top supernatant, which is the LPS solution, and the LPS concentration is 150 μg / mL measured by sulfuric acid-phenol method;

[0094] (3) Synthetic DMON

[0095] Under stirring conditions, 68 mg of TEA was first added to 25 mL of water and gently stirred in an 80 °C oil bath for 0.5 h to ensure that TEA was evenly dispersed and fully dissolved; 380 mg of CTAB and 168 mg of NaSal were then added to the above solution and continued to be stirred in an 80 °C oil bath for 1 h to allow CTAB and NaSal to fully dissolve and form a stable template structure; then, 2 mL of TEOS and 1.6 mL of BTEE were mixed and added to the above water-CTAB-NaSal-TEA, and continued to be stirred in an 80 °C oil bath for 1 h. After the reaction was completed, the reaction product was quenched with HCl and methanol solution (V HCl : V 甲醇 =1:9) was refluxed and extracted at 60 °C for 6 h, and extracted 3 times in total to remove the template, and finally vacuum dried at room temperature overnight to obtain DMON powder;

[0096] (4) Preparation of DMON-NH2

[0097] The DMON powder obtained in step (3) was mixed with anhydrous ethanol to obtain a DMON ethanol solution with a concentration of 10 mg / mL; 5 mL of the DMON ethanol solution was mixed with 5 μL of APTES, and the mixture was stirred at 500 rpm for 12 h at room temperature to ensure that the reaction was fully carried out; after the reaction was completed, the reaction product was centrifuged at 10,000 rpm, and the precipitate was washed with an appropriate amount of water to remove unreacted APTES. The washed reaction product was vacuum dried at room temperature for 12 h to obtain positively charged amino-modified DMON-NH2;

[0098] (5) Preparation of LPS@DMON@OMV

[0099] The DMON-NH2 prepared in step (4) was mixed with water to obtain a DMON-NH2 solution with a concentration of 10 mg / mL; 500 μL of the LPS solution (150 μg / mL) prepared in step (3) and 2 mL of the DMON-NH2 solution (10 mg / mL) were mixed, and the mixture was stirred at 500 rpm for 12 h at room temperature to allow DMON to load LPS through electrostatic interaction; after the reaction was completed, the reaction product was centrifuged at 10,000 rpm and washed with an appropriate amount of water. The washed reaction product was re-dissolved with 2 mL of 1×PBS buffer solution to obtain an LPS@DMON solution; 500 μL of the obtained LPS@DMON solution was mixed with 500 μL of The OMV solution (concentration of 1 mg / mL) was mixed at 4°C, extruded back and forth through a 200 nm polycarbonate film 10 times using an Avanti microliposome extruder, and finally centrifuged at 8000 rpm for 10 min at 4°C to separate the unloaded OMVs to obtain the Helicobacter pylori outer membrane vesicle-based nanovaccine (LPS@DMON@OMV).

[0100] Comparative Example 1

[0101] (1)-(4) Same as Example 1;

[0102] (5) Preparation of LPS@DMON@OMV

[0103] The amount of LPS solution used was 1000 μL, which was twice that of Example 1. Other operations were the same as those of Example 1.

[0104] Comparative Example 2

[0105] (1)-(4) Same as Example 1;

[0106] (5) Preparation of LPS@DMON@OMV

[0107] The amount of LPS solution used was 167 μL, which was 1 / 3 of that in Example 1. Other operations were the same as in Example 1.

[0108] Comparative Example 3

[0109] (1)-(4) Same as Example 1;

[0110] (5) Preparation of LPS@DMON@OMV

[0111] The amount of OMV solution (concentration of 2 mg / mL) used was 500 μL, which was twice that of Example 1. Other operations were the same as Example 1.

[0112] Comparative Example 4

[0113] (1)-(4) Same as Example 1;

[0114] (5) Preparation of LPS@DMON@OMV

[0115] The amount of OMV solution (concentration of 0.5 mg / mL) used is 500 μL, which is 1 / 2 of that in Example 1. Other operations are the same as in Example 1.

[0116] Comparative Example 5

[0117] (1)-(4) Same as Example 1;

[0118] (5) Preparation of LPS@DMON@OMV

[0119] The amount of DMON-NH2 (concentration of 20 mg / mL) used was 2 mL, which was twice that of Example 1. Other operations were the same as those of Example 1.

[0120] Effect Example 1

[0121] The usage (mass) ratios of LPS, OMV and DMON-NH2 in Example 1 and Comparative Examples 1-5 are shown in Table 1.

[0122] Table 1 The ratio of LPS, OMV and DMON-NH2 used in Example 1 and Comparative Examples 1-5

[0123] Comparative group <![CDATA[LPS:OMV:DMON-NH2]]> Example 1 0.75:20:200 Comparative Example 1 1.5:20:200 Comparative Example 2 0.25:20:200 Comparative Example 3 0.75:40:200 Comparative Example 4 0.75:10:200 Comparative Example 5 0.75:20:400

[0124] Among them, compared with Example 1, the increase in the amount of LPS used in Comparative Example 1 will lead to an increase in the toxicity of the final product, and the cell survival rate of the prepared nanovaccine in vitro experiment is greatly reduced. Among them, when the OMV experimental concentration is 20 μg / mL, the CCK-8 cytotoxicity 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 amount of LPS used in Comparative Example 2 is insufficient, which will result in a lower level of immune cytokines in the in vitro experiment, that is, a decrease in the immune effect. When the OMV experimental concentration is 20 μg / mL, the results of the quantitative detection of in vitro immune cytokines show (the specific detection method refers to Example 2): the levels of IFN-γ, IL-4 and IL-17A in the LPS@DMON@OMV group of Comparative Example 2 are not significantly different from those in the DMON@OMV group, and are significantly lower than those in Example 1;

[0126] Compared with Example 1, the amount of OMV used in Comparative Example 3 increased, and the protein loading rate was limitedly improved. Among them, compared with Example 1, the OMV protein loading rate was increased by 13%, which would cause a waste of antigen cost and greatly increase the raw material cost;

[0127] Compared with Example 1, the OMV in Comparative Example 4 is insufficient, resulting in unsaturated carriers, that is, the protein loading rate is low. Compared with Example 1, the protein loading rate (for specific detection methods, refer to Effect Example 2) is reduced by 60%, and the utilization rate is low, which affects the immune effect;

[0128] Compared with Example 1, the excessive amount of DMON-NH2 in Comparative Example 5 may cause blockage of the extruder, resulting in a decrease in the extrusion throughput rate.

[0129] In summary, the mass ratio of LPS, OMV, and DMON-NH2 was 0.75:20:200, which could obtain a relatively stable protein and LPS loading ratio.

[0130] Comparative Example 6

[0131] (1) Prepare OMV solution according to Example 1;

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

[0133] (3) 500 μL of DMON aqueous solution (10 mg / mL) and 500 μL of OMV solution (1 mg / mL) were mixed at 4°C and extruded 10 times through a 200 nm polycarbonate film using an Avanti microliposome extruder. Finally, the unloaded OMVs were separated by centrifugation at 8000 rpm for 10 min at 4°C to obtain DMON@OMV.

[0134] Effect Example 2

[0135] 1. Test methods

[0136] The following tests were conducted using OMV, LPS, DMON, DMON-NH2, LPS@DMON and LPS@DMON@OMV prepared in Example 1 and DMON@OMV prepared in Comparative Example 6 as the subjects:

[0137] 1.1 Sample test characterization

[0138] 1.1.1 Morphology and Zeta potential

[0139] (1) Transmission electron microscopy (TEM) was used to observe the morphology of OMV, DMON and LPS@DMON@OMV prepared in Example 1. The samples were placed on a 200-mesh copper grid and stained with 2% phosphotungstic acid negative staining solution (Legen Biotechnology, Catalog No.: DZ0035). Then, TEM images were obtained at 80 kV.

[0140] (2) The surface potentials of OMV, LPS, DMON, DMON-NH2, LPS@DMON, DMON@OMV, and LPS@DMON@OMV were measured using dynamic light scattering (DLS).

[0141] 1.1.2 Membrane protein evaluation

[0142] (1) The total protein content of OMVs 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 conventional methods.

[0144] 1.1.3 LPS concentration determination

[0145] The sulfuric acid-phenol method (Wang Xitong, Yang Jiaxiang, Wang Aijia, et al. Rapid determination of watermelon rind polysaccharide content by trace 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.) was used to determine the amount of LPS. 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. Within a certain concentration range, the absorbance at 490 nm is linearly related to the polysaccharide content. A standard curve is drawn 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 testing

[0147] 1.2.1 Cytotoxicity assay

[0148] Mouse macrophages RAW264.7 (Cell Bank of Chinese Academy of Sciences, Shanghai, China) in logarithmic growth phase were cultured at 5 × 10 3The density of cells / well was inoculated in a 96-well plate (100 μL / well). After the cells were completely attached to the wall, DMEM complete medium containing LPS@DMON@OMV was added (OMV final concentration gradient: 0, 10, 20, 40, 60, 80 μg / mL), and the cell-free medium was used as a blank control. After each group of samples was cultured at 37°C and 5% CO2 for 24 hours, 10 μL of CCK-8 working solution was added to each well and incubated for another 1 hour in the dark. The absorbance of each well was measured at a wavelength of 450 nm using an enzyme reader to calculate the relative cell survival rate.

[0149] 1.2.2 Analysis of cell phagocytosis effect

[0150] According to the instruction manual, the exosome fluorescent dye DIR (Umibio, Cat. No. UR21017) was used to label OMV and LPS@DMON@OMV nanoparticles. RAW264.7 mouse macrophages with good growth status were taken at 5×10 5 Cells / well were inoculated in 6-well plates. After the cells adhered to the wall, the above-mentioned DIR fluorescently labeled OMVs and LPS@DMON@OMV nanoparticles (OMV final concentration 20μg / mL) were added respectively. Cells added with PBS were used as the control group. After 6 hours of co-culture, the culture supernatant was removed and the cells were washed 3 times with PBS to remove the uninternalized nanoparticles. An appropriate amount of 4% paraformaldehyde fixative (Biosharp, catalog number BL539A-1) was added to fix the cells for 20 minutes, washed twice with PBS, and then the cell nuclei were stained with DAPI staining solution (working concentration 1μg / mL) for 15 minutes. After washing with PBS to remove the residual dye, multi-channel fluorescence imaging was performed using a confocal laser scanning microscope (CLSM). DIR-labeled nanoparticles showed red fluorescence (excitation / emission wavelength: 748 / 780nm), and DAPI nuclear staining showed blue fluorescence (excitation / emission wavelength: 358 / 461nm).

[0151] 1.2.3 Quantitative detection of immune cytokines

[0152] Mouse macrophage RAW264.7 cells were cultured at 5 × 10 3Cells were inoculated in 96-well plates at a density of 10 cells / well. The experimental groups were treated with OMV, DMON@OMV and LPS@DMON@OMV (n=6). The final concentration of antigen OMV in each group was 20 μg / mL. Cells added with PBS were used as the control group. After 24 h of culture, the cells were centrifuged at 1200×g for 10 min at 4°C to remove cell debris and take the supernatant. The secretion levels of IFN-γ, IL-4 and IL-17A were quantitatively detected according to the instructions of the ELISA kit. The mouse interferon gamma (IFN-γ) kit (Cat. No. RX203097M), mouse interleukin 4 (IL-4) kit (Cat. No. RX203051M), and mouse interleukin 17A (IL-17A) kit (Cat. No. RX203066M) were all purchased from Ruixin Biotechnology. The standard curve drawing and concentration calculation strictly followed the technical specifications of the kit.

[0153] 1.3 Animal Immunization Experiment Evaluation

[0154] 1.3.1 Immunization Procedure

[0155] Six-week-old female C57BL / 6 mice (weight 16-22 g, purchased from Guangdong Medical Experimental Animal Center) were selected and adaptively raised in an SPF environment for 14 days, and then randomly divided into 4 groups (n=4 / group), named as: ①PBS; ②OMV; ③DMON@OMV; ④LPS@DMON@OMV; except for the PBS group, each group was given the corresponding antigen preparation solution by oral gavage on the 1st, 7th and 14th days, where the volume of each gavage of the antigen preparation solution was 100 μL, and the amount of the antigen preparation was 40 μg (calculated as 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, incubated at 37°C for 1 h, then stored at 4°C overnight, centrifuged at 3000 g for 15 min, and the supernatant serum was aliquoted and stored at -80°C for later use;

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

[0158] (3) Gastric tissue homogenization: The mice were killed and the gastric tissue was removed under sterile conditions. The gastric tissue was rinsed with pre-cooled PBS to remove residual blood, and the gastric tissue was cut into pieces after weighing. The gastric tissue was homogenized in PBS containing 0.5 mM PMSF protease inhibitor (Biyuntian, Catalog No. ST506) at a weight-to-volume ratio of 1:9, and the mixture was fully ground on ice. Finally, the homogenate was centrifuged at 5000 × g for 10 min, the supernatant was collected, and stored at -80°C for later use.

[0159] (4) Isolation of spleen cells: The mice were killed, and the spleens were removed under sterile conditions and placed in a 6 cm cell culture dish containing 5 mL of 1640 culture medium. The cells were separated by mechanical separation. The spleen tissue was pushed through a 40 μm cell sieve using the plunger of a syringe. The sieve was then thoroughly rinsed with cell suspension. The cells were centrifuged at 600 × g for 5 min at 4 °C and the supernatant was discarded. The cells were resuspended in 1 mL of 1× red blood cell lysis buffer (Biyuntian, Catalog No. C3702) and incubated at room temperature for 5 min, with occasional gentle shaking or rotation. 3 mL of 1640 culture medium was added to terminate the lysis reaction. The cells were centrifuged at 600 × g for 5 min, the supernatant was discarded, and the cell density was adjusted to 1 × 10 7 cells / mL;

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

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

[0162] (1) Coating antigen: Dilute Helicobacter pylori SS1 to 10 using carbonate buffer (pH 9.6). 8 CFU / mL, 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 three times with PBST, add 300 μL of PBS solution containing 3% (mass percentage) skimmed milk powder to each well, and block at 37°C for 1 h;

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

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

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

[0167] (6) Titer calculation: Judgment threshold = negative control OD mean × 2.1. Sample titer is defined as the reciprocal of the highest dilution that produces a threshold signal. Draw the antibody titer curve with the dilution multiple as the horizontal axis and the OD value as the vertical axis.

[0168] 1.3.3 Evaluation of anti-Helicobacter pylori antibody specificity

[0169] Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, and Acinetobacter baumannii ATCC 19606 were used to specifically analyze the anti-Helicobacter pylori antibodies in the serum of the LPS@DMON@OMV group prepared in step 1.3.1 (1). PBS was set as the negative control. The specific steps were referred to step 1.3.2. The antibody titer curve was plotted with the dilution multiple as the horizontal axis and the OD value as the vertical axis.

[0170] 1.3.4 Determination of mucosal immune IgA levels

[0171] The IgA levels of 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 operating instructions of the ELISA kit (mouse immunoglobulin A (IgA) kit, Ruixin Bio, catalog number RX202739M). ​​The standard curve drawing and concentration calculation strictly followed the technical specifications of the kit.

[0172] 1.3.5 Analysis of spleen cell immune factors

[0173] The spleen cells prepared in step 1.3.1 (4) were plated at 1×10 7The cells / well density was inoculated in a 24-well plate, 500 μL / well, and OMV, DMON@OMV and LPS@DMON@OMV were added to enhance the stimulation of mouse spleen cells. The final concentration of antigen OMV was 10 μg / mL. The wells without antigen were used as controls. After 24 hours of culture, the cells were centrifuged at 1200×g for 10 minutes at 4°C to remove cell debris and take the supernatant. The levels of IFN-γ, IL-4 and IL-17A in the cell supernatant were quantitatively detected according to the operating instructions of the ELISA kit (same as 1.2.3). The standard curve drawing and concentration calculation strictly followed the technical specifications of the kit.

[0174] 1.3.6 Safety evaluation

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

[0176] 2. Test results

[0177] 2.1 Sample test characterization results

[0178] 2.1.1 TEM and Zeta potential results

[0179] Figure 1 TEM results of OMV, DMON, and LPS@DMON@OMV. Figure 1 It can be seen that a film with a diameter of about 150-200 nm is formed on the surface of LPS@DMON@OMV particles.

[0180] Figure 2 It is the surface Zeta potential of each component and intermediate in the process of synthesizing nano-vaccine to verify the stable generation of vaccine. Figure 2 It can be seen that after loading LPS, the value of DMON-NH2 (surface modified amino group) 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 shows that the coating of OMV is successful, because after the coating is completed, the electronegativity of LPS@DMON@OMV increases to −27.9±0.9 mV due to the electronegativity of OMV.

[0181] 2.1.2 Protein analysis and LPS concentration determination

[0182] Figure 3The SDS-PAGE results of the protein components of OMV, DMON, DMON@OMV, and LPS@DMON@OMV were further verified by gel electrophoresis, and the results showed that the membrane protein composition of the H. pylori outer membrane vesicles was preserved throughout the preparation process. No protein was detected in DMON nanoparticles, and the protein composition content of LPS@DMON@OMV was not significantly different from that of DMON@OMV, indicating that LPS had no effect on the loading of OMV.

[0183] In addition, the Bradford protein Assay Kit (BCA) was used to determine the total protein content of OMVs on LPS@DMON@OMV nanoparticles. The results showed that the OMV protein loading ratio (the weight ratio of the fixed protein on the nanoparticles) was approximately 38.2±0.5 μg / mg. The sulfuric acid-phenol method determined that the loading amount of LPS in LPS@DMON@OMV nanoparticles was approximately 24.8±3.7 μg / mg.

[0184] 2.2 In vitro cell experiment results

[0185] 2.2.1 Cytotoxicity assay

[0186] The effect of LPS@DMON@OMV nanoparticles on the proliferation of macrophages was studied by CCK-8. 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 lower than 40 μg / mL.

[0187] 2.2.2 Cellular uptake results

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

[0189] 2.2.3 Effect of RAW264.7 factor secretion in mouse macrophages

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

[0191] 2.3 Animal immune effect study

[0192] 2.3.1 Serum anti-Helicobacter pylori IgG titer

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

[0194] 2.3.2 Specificity of anti-Helicobacter pylori antibodies

[0195] like Figure 8 As shown in the figure, 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 higher specificity.

[0196] 2.3.3 Mucosal IgA Levels

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

[0198] 2.3.4 Levels of spleen cell immune factors

[0199] IFN-γ reflects Th1 immune response, IL-4 reflects Th2 immune response, and IL-17A is an indicator of Th17 immune response. Fig.10This is the analysis of the results of IFN-γ, IL-4 and IL-17A in the spleen cells of mice after immunization. It can be seen from the figure that the secretion levels of cytokines IFN-γ, IL-4 and IL-17A after immunization are 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 three control groups, indicating that the vaccine can produce an effective mixed Th1 / Th2 / Th17 immune response.

[0200] 2.3.5 Effects of immunity on blood immune cells

[0201] Blood routine analysis results ( Fig.11 ) showed that the levels of white blood cells (WBC), lymphocytes (LYM), neutrophils (NEU) and monocytes (MON) in mice were significantly increased after LPS@DMON@OMV immunization, while there was no significant difference between the OMV group and the DMON@OMV group, indicating that the cellular immunity level of animals was significantly enhanced after LPS@DMON@OMV immunization.

[0202] 2.3.6 Safety evaluation

[0203] Fig.12 The blood routine analysis was to check the red blood cell content (RBC), hemoglobin content (HGB) and platelet content (PLT). The results showed that there was no significant change in the levels of these indicators.

[0204] In addition, the mouse heart, liver, spleen, lung, and kidney were stained with HE. Fig.13 As can be seen from the figure, compared with the PBS control group, there was no lesion in the visceral tissues of the immunized mice, and there was no significant difference in the organs. This indicates that the results support the safety of LPS@DMON@OMV nanovaccine.

[0205] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are 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.

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 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.

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 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.

7. 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.

8. 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.

9. 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 8.

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

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