System and method for preparing outer membrane vesicles with guaranteed immune effects

Abstract

Provided are a system and method for preparing outer membrane vesicles with guaranteed immune effects. The system comprises a strain acquisition module, a strain screening module, an outer membrane vesicle yield promotion module, an outer membrane vesicle extraction module, an outer membrane vesicle purification module, an immune efficacy evaluation module, and a yield evaluation module, wherein the strain acquisition module is connected to the strain screening module, the outer membrane vesicle yield promotion module, and the outer membrane vesicle extraction module, respectively. By means of a genetic modification operation, the alr gene of Brucella is knocked out, which in turn improves the yield and immune efficacy of the outer membrane vesicles. Moreover, by means of strictly controlling the Brucella culture conditions, an inducing environment is established to induce Brucella to secrete outer membrane vesicles, thereby further improving the yield of outer membrane vesicles, and in turn obtaining outer membrane vesicles with a high yield and good immune effects, which meets the requirements of large-scale vaccine production, and provides a high-quality vaccine adjuvant for the research into, development of and application of Brucella vaccines.

Description

A preparation system and method for outer membrane vesicles ensuring immune effect Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a system and method for preparing outer membrane vesicles capable of ensuring immune effects. Background Art

[0002] Brucella outer membrane vesicles are a kind of structure released into the extracellular space by Brucella during growth. They have a double membrane and are composed of lipoproteins, outer membrane proteins, lipopolysaccharides and some periplasmic components. They contain the immunogenic proteins necessary for the bacterium and a large amount of pathogen-related pattern molecules. They can effectively induce the body's nonspecific immunity and have potential value as a vaccine adjuvant. However, the existing Brucella outer membrane vesicles have weak immune efficacy, resulting in unsatisfactory vaccine effects, and because the preparation method is less efficient, it is difficult to meet the needs of large-scale production and application. This makes the effect and output of outer membrane vesicles unable to meet the needs of clinical and vaccine research and development, and it is impossible to give full play to its potential as a vaccine adjuvant. Therefore, a preparation system and method for outer membrane vesicles that ensure immune effect are proposed.

[0003] Summary of the Invention

[0004] The purpose of the present invention is to provide a system and method for preparing outer membrane vesicles that ensure immune effects, so as to solve the problems raised in the above background technology.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solutions: a system for preparing outer membrane vesicles that ensures immune effects, comprising a strain acquisition module, a strain screening module, an outer membrane vesicle yield promotion module, an outer membrane vesicle extraction module, an outer membrane vesicle purification module, an immune efficacy evaluation module and a yield evaluation module, wherein the strain acquisition module is respectively connected to the strain screening module, the outer membrane vesicle yield promotion module and the outer membrane vesicle extraction module, the outer membrane vesicle extraction module is respectively connected to the outer membrane vesicle purification module, and the outer membrane vesicle purification module is respectively connected to the immune efficacy evaluation module and the yield evaluation module.

[0006] Preferably, the strain acquisition module includes a recombinant vector construction submodule, a recombinant vector introduction submodule, a strain culture submodule and a culture condition control submodule, and the recombinant vector construction submodule is connected to the recombinant vector introduction submodule, and the strain culture submodule is connected to the culture condition control submodule. The recombinant vector construction submodule is used for the recombinant vector, the recombinant vector introduction submodule is used to import the recombinant vector into the competent cells of Brucella, the strain culture submodule is used to culture Brucella, and the culture condition control submodule is used to control the culture conditions of Brucella.

[0007] Preferably, the culture condition control submodule includes a culture medium component control unit, a culture temperature control unit, an oxygen concentration control unit, a pH control unit and a culture time control unit. The culture medium component control unit is used to control the nutrient composition and ion concentration of the culture medium, the culture temperature control unit is used to control the temperature of the culture environment, the oxygen concentration control unit is used to control the oxygen concentration of the culture environment, the pH control unit is used to control the pH of the culture environment, and the culture time control unit is used to control the culture time.

[0008] Preferably, the strain screening module includes a PCR amplification submodule, a gel imaging submodule and a gene identification submodule. The PCR amplification submodule is used to amplify gene deletion fragments, the gel imaging submodule is used to observe the electrophoresis results of PCR products and screen the target strains, and the gene identification submodule is used to perform gene sequencing on the screened target strains.

[0009] Preferably, the outer membrane vesicle production promotion module includes an artificial inducer module, an induction environment creation submodule and an induction time control submodule, and the induction environment creation submodule is connected to the artificial inducer module and the induction time control submodule respectively. The artificial inducer module is used to set the induction conditions, the induction environment creation submodule is used to execute the conditions set by the artificial inducer module and create an induction environment, and the induction time control submodule is used to control the induction time.

[0010] Preferably, the artificial inducer module includes a chemical induction unit and a physical induction unit. The chemical induction unit is used to set chemical induction conditions, including the composition and dosage of the inducer, and the physical induction unit is used to set physical induction conditions.

[0011] Preferably, the outer membrane vesicle extraction module includes an ultrasonic crushing treatment submodule and a differential centrifugation treatment submodule, and the ultrasonic crushing treatment submodule is connected to the differential centrifugation treatment submodule, the ultrasonic crushing treatment submodule is used for ultrasonic treatment of Brucella, and the differential centrifugation treatment submodule is used to extract outer membrane vesicles.

[0012] Preferably, the immune efficacy evaluation module includes a cell level evaluation submodule and an animal level evaluation submodule. The cell level evaluation submodule is used to evaluate the immune efficacy of outer membrane vesicles at the cell level, and the animal level evaluation submodule is used to evaluate the immune efficacy of outer membrane vesicles at the animal level.

[0013] A method for preparing outer membrane vesicles with guaranteed immune effects, comprising the steps of: first, obtaining a strain; second, culturing the strain; third, promoting outer membrane vesicle secretion; fourth, extracting and purifying the outer membrane vesicles; and fifth, evaluating immune efficacy and yield.

[0014] Wherein in the above-mentioned step 1, utilize the recombinant vector construction submodule to perform gene modification operation, then construct a recombinant vector, utilize the recombinant vector import submodule again to import the recombinant vector into the competent cells of Brucella, and through the mode of homologous recombination, make the gene on the chromosome be replaced, obtain genetically modified Brucella;

[0015] In the above step 2, the Brucella obtained in step 1 is cultured using the strain culture submodule, and then the Brucella with successful genetic modification is screened out using the strain screening module, and cultured to obtain the target bacteria;

[0016] In the above step 3, the outer membrane vesicle production promotion module is used to induce the target bacteria in step 2 to secrete outer membrane vesicles;

[0017] In the above step 4, the target bacteria after the induction treatment in step 3 are collected, the outer membrane vesicles are extracted using the outer membrane vesicle extraction module, and the extracted outer membrane vesicles are purified using the outer membrane vesicle purification module;

[0018] In the above step five, the outer membrane vesicles purified in step four are collected, the immune efficacy of the outer membrane vesicles is evaluated using the immune efficacy evaluation module, and the yield of the outer membrane vesicles is evaluated using the yield evaluation module.

[0019] Preferably, in the step 1, in the gene modification operation, the gene knocked out is the alr gene.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention knocks out the alr gene of Brucella through genetic modification operation, thereby increasing the yield and immune efficacy of outer membrane vesicles, and by strictly controlling the Brucella culture conditions, creating an inducing environment to induce Brucella to secrete outer membrane vesicles, thereby further increasing the yield of outer membrane vesicles, thereby obtaining high-yield outer membrane vesicles with good immune effects to meet the needs of large-scale vaccine production, and providing high-quality vaccine adjuvants for the research and development and application of Brucella vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG1 is a block diagram of the system structure of the present invention;

[0022] FIG2 is a block diagram of the bacterial strain acquisition module of the present invention;

[0023] FIG3 is a structural block diagram of an outer membrane vesicle production promotion module of the present invention;

[0024] FIG4 is a flow chart of the steps of the present invention;

[0025] FIG5 is a flow chart of the method of the present invention.

[0026] In the figure: 1. Strain acquisition module; 11. Recombinant vector construction submodule; 12. Recombinant vector introduction submodule; 13. Strain culture submodule; 14. Culture condition control submodule; 141. Culture medium component control unit; 142. Culture temperature control unit; 143. Oxygen concentration control unit; 144. pH control unit; 145. Culture time control unit; 2. Strain screening module; 21. PCR amplification submodule; 22. Gel imaging submodule; 23. Gene identification submodule; 3. Outer membrane vesicle production promotion module; 31. Artificial inducer submodule; 311. Chemical induction unit; 312. Physical induction unit; 32. Induction environment creation submodule; 33. Induction time control submodule; 4. Outer membrane vesicle extraction module; 41. Ultrasonic fragmentation treatment submodule; 42. Differential centrifugation treatment submodule; 5. Outer membrane vesicle purification module; 6. Immune efficacy evaluation module; 61. Cell level evaluation submodule; 62. Animal level evaluation submodule; 7. Production evaluation module. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] Please refer to Figures 1 to 3. An embodiment of the present invention is: a system for preparing outer membrane vesicles to ensure immune effect, including a strain acquisition module 1, a strain screening module 2, an outer membrane vesicle yield promotion module 3, an outer membrane vesicle extraction module 4, an outer membrane vesicle purification module 5, an immune efficacy evaluation module 6 and a yield evaluation module 7. The strain acquisition module 1 is connected to the strain screening module 2, the outer membrane vesicle yield promotion module 3 and the outer membrane vesicle extraction module 4 respectively, the outer membrane vesicle extraction module 4 is connected to the outer membrane vesicle purification module 5, the outer membrane vesicle purification module 5 is connected to the immune efficacy evaluation module 6 and the yield evaluation module 7 respectively, the strain acquisition module Block 1 is used to obtain the target strain, the strain screening module 2 is used to screen the strains with successful gene modification, the outer membrane vesicle production promotion module 3 is used to promote the target strain to secrete outer membrane vesicles, the outer membrane vesicle extraction module 4 is used to extract outer membrane vesicles, the outer membrane vesicle purification module 5 is used to purify outer membrane vesicles, the immune efficacy evaluation module 6 is used to evaluate the immune efficacy of outer membrane vesicles, and the production evaluation module 7 is used to evaluate the production of outer membrane vesicles; the strain acquisition module 1 includes a recombinant vector construction submodule 11, a recombinant vector introduction submodule 12, a strain culture submodule 13 and a culture condition control submodule 14, and the recombinant vector construction submodule 11 is connected to the recombinant vector introduction submodule 12. Then, the strain culture submodule 13 is connected to the culture condition control submodule 14, the recombinant vector construction submodule 11 is used for the recombinant vector, the recombinant vector introduction submodule 12 is used to introduce the recombinant vector into the competent cells of Brucella, the strain culture submodule 13 is used to culture Brucella, and the culture condition control submodule 14 is used to control the culture conditions of Brucella; the culture condition control submodule 14 includes a culture medium component control unit 141, a culture temperature control unit 142, an oxygen concentration control unit 143, a pH control unit 144 and a culture time control unit 145, and the culture medium component control unit 141 is used to control the nutrition of the culture medium The composition and ion concentration of the culture environment are controlled by the culture temperature control unit 142, the oxygen concentration control unit 143 is used to control the oxygen concentration of the culture environment, the pH control unit 144 is used to control the pH of the culture environment, and the culture time control unit 145 is used to control the culture time; the strain screening module 2 includes a PCR amplification submodule 21, a gel imaging submodule 22 and a gene identification submodule 23, the PCR amplification submodule 21 is used to amplify gene deletion fragments, the gel imaging submodule 22 is used to observe the electrophoresis results of the PCR products and screen the target strains, and the gene identification submodule 23 is used to perform gene sequencing on the screened target strains;The outer membrane vesicle production promotion module 3 includes an artificial induction submodule 31, an induction environment creation submodule 32 and an induction time control submodule 33, and the induction environment creation submodule 32 is connected to the artificial induction submodule 31 and the induction time control submodule 33 respectively. The artificial induction submodule 31 is used to set the induction conditions, the induction environment creation submodule 32 is used to execute the conditions set by the artificial induction submodule 31 and create an induction environment, and the induction time control submodule 33 is used to control the induction time; the artificial induction submodule 31 includes a chemical induction unit 311 and a physical induction unit 312. The chemical induction unit 311 is used to set the chemical induction conditions, including the composition of the inducer and the use of the inducer. The physical induction unit 312 is used to set the physical induction conditions; the outer membrane vesicle extraction module 4 includes an ultrasonic fragmentation processing submodule 41 and a differential centrifugation processing submodule 42, and the ultrasonic fragmentation processing submodule 41 and the differential centrifugation processing submodule 42 are connected. The ultrasonic fragmentation processing submodule 41 is used to ultrasonically treat Brucella, and the differential centrifugation processing submodule 42 is used to extract outer membrane vesicles; the immune efficacy evaluation module 6 includes a cell-level evaluation submodule 61 and an animal-level evaluation submodule 62. The cell-level evaluation submodule 61 is used to evaluate the immune efficacy of outer membrane vesicles at the cell level, and the animal-level evaluation submodule 62 is used to evaluate the immune efficacy of outer membrane vesicles at the animal level.

[0029] Referring to Figures 4 and 5 , an embodiment of the present invention provides a method for preparing outer membrane vesicles with guaranteed immune effects, comprising the steps of: 1. obtaining a strain; 2. culturing the strain; 3. promoting outer membrane vesicle secretion; 4. extracting and purifying the outer membrane vesicles; and 5. evaluating the immune efficacy and yield.

[0030] Wherein in the above-mentioned steps one, utilize recombinant vector construction submodule 11 to perform genetic modification operation, then construct recombinant vector, reutilize recombinant vector import submodule 12 that recombinant vector is imported into the competent cell of Brucella, by the mode of homologous recombination, make the gene on chromosome be replaced, obtain the Brucella after genetic modification; Wherein, in the genetic modification operation, the gene knocked out is alr gene;

[0031] Wherein in the above step 2, the Brucella obtained in step 1 is cultured using the strain culture submodule 13, and then the Brucella with successful genetic modification is screened out using the strain screening module 2, and is cultured to obtain the target bacterium;

[0032] In the above step 3, the outer membrane vesicle production promotion module 3 is used to induce the target bacteria in step 2 to secrete outer membrane vesicles;

[0033] In the above step 4, the target bacteria after the induction treatment in step 3 are collected, the outer membrane vesicles are extracted using the outer membrane vesicle extraction module 4, and the extracted outer membrane vesicles are purified using the outer membrane vesicle purification module 5;

[0034] In the above step 5, the outer membrane vesicles purified in step 4 are collected, and the immune efficacy of the outer membrane vesicles is evaluated using the immune efficacy evaluation module 6, and the yield of the outer membrane vesicles is evaluated using the yield evaluation module 7.

[0035] Based on the above, the advantage of the present invention is that, when the invention is used, the recombinant vector construction submodule 11 in the bacterial strain acquisition module 1 is first utilized to perform genetic modification operation, the alr gene of Brucella is knocked out, and a recombinant vector is constructed, and then the recombinant vector is imported into the competent cell of Brucella by the recombinant vector import submodule 12, and the gene on the chromosome is replaced by homologous recombination to obtain the Brucella after genetic modification; the bacterial strain culture submodule 13 is utilized to cultivate the Brucella obtained, and then the bacterial strain screening module 2 is utilized to screen out the Brucella that is genetically modified successfully, and it is cultivated to obtain the target thalline, and in the culturing process, the culture condition control submodule 12 is utilized to control the Brucella after genetic modification. Module 14 controls the culture environment in the optimal conditions, specifically: using the culture medium component control unit 141 to control the nutrient composition and ion concentration of the culture medium, using the culture temperature control unit 142 to control the temperature of the culture environment, using the oxygen concentration control unit 143 to control the oxygen concentration of the culture environment, using the pH control unit 144 to control the pH of the culture environment, and using the culture time control unit 145 to control the culture time; using the strain screening module 2 to screen specifically: using the PCR amplification submodule 21 to perform PCR amplification, using the gel imaging submodule 22 to observe the electrophoresis results of the PCR product, screening out the Brucella that has successfully been genetically modified, and then using the gene identification submodule Module 23 performs gene sequencing to further identify whether the gene modification is successful; the outer membrane vesicle production promotion module 3 is used to induce the target bacteria to secrete outer membrane vesicles, specifically: the chemical induction unit 311 is used to set the chemical induction conditions, including the composition and dosage of the inducer, the physical induction unit 312 is used to set the physical induction conditions, the induction environment creation submodule 32 is used to execute the conditions set by the artificial induction submodule 31 to create an induction environment, thereby promoting the target bacteria to secrete a large amount of outer membrane vesicles, and the induction time control submodule 33 is used to control the induction process within the optimal time; the target bacteria after the induced treatment are collected and the ultrasonic fragmentation treatment submodule 4 in the outer membrane vesicle extraction module 4 is used. 1. The target bacteria are crushed, and outer membrane vesicles are obtained by centrifugation using the differential centrifugation submodule 42. The extracted outer membrane vesicles are then subjected to ultrafiltration purification by the outer membrane vesicle purification module 5. The purified outer membrane vesicles are collected, and the immune efficacy of the outer membrane vesicles is evaluated at the cellular level and the animal level using the cell level evaluation submodule 61 and the animal level evaluation submodule 62 of the immune efficacy evaluation module 6, respectively. The yield of the outer membrane vesicles is evaluated using the yield evaluation module 7. Specifically, the outer membrane vesicles are used as vaccine adjuvants, added together with Brucella during cell infection, and changes in cytokines and immune-related proteins are detected, thereby achieving the immune efficacy of the outer membrane vesicles at the cellular level.Using outer membrane vesicles as vaccine adjuvants, animal immunization studies were conducted to assess the immune efficacy of outer membrane vesicles at the animal level. Flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay), and Western blot were used to detect the number of immune CD4+, CD8+, and Treg cells, as well as changes in serum IgG, IgG1, IgG2a, cytokines IL-6, IL-10, IFN-γ, TNF-α, and related proteins. HE staining was used to analyze the white pulp, red pulp, and germinal centers of the spleen.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A system for preparing outer membrane vesicles that ensures immune effects, comprising a strain acquisition module (1), a strain screening module (2), an outer membrane vesicle yield promotion module (3), an outer membrane vesicle extraction module (4), an outer membrane vesicle purification module (5), an immune efficacy evaluation module (6) and a yield evaluation module (7), characterized in that: The strain acquisition module (1) is connected to the strain screening module (2), the outer membrane vesicle production promotion module (3) and the outer membrane vesicle extraction module (4), respectively; the outer membrane vesicle extraction module (4) is connected to the outer membrane vesicle purification module (5), and the outer membrane vesicle purification module (5) is connected to the immune efficacy evaluation module (6) and the production evaluation module (7).

2. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 1, characterized in that: The strain acquisition module (1) includes a recombinant vector construction submodule (11), a recombinant vector introduction submodule (12), a strain culture submodule (13) and a culture condition control submodule (14), and the recombinant vector construction submodule (11) is connected to the recombinant vector introduction submodule (12), and the strain culture submodule (13) is connected to the culture condition control submodule (14). The recombinant vector construction submodule (11) is used for recombinant vectors, the recombinant vector introduction submodule (12) is used for introducing the recombinant vectors into competent cells of Brucella, the strain culture submodule (13) is used for culturing Brucella, and the culture condition control submodule (14) is used for controlling the culture conditions of Brucella.

3. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 2, characterized in that: The culture condition control submodule (14) includes a culture medium component control unit (141), a culture temperature control unit (142), an oxygen concentration control unit (143), a pH control unit (144) and a culture time control unit (145). The culture medium component control unit (141) is used to control the nutrient components and ion concentration of the culture medium, the culture temperature control unit (142) is used to control the temperature of the culture environment, the oxygen concentration control unit (143) is used to control the oxygen concentration of the culture environment, the pH control unit (144) is used to control the pH of the culture environment, and the culture time control unit (145) is used to control the culture time.

4. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 1, characterized in that: The strain screening module (2) comprises a PCR amplification submodule (21), a gel imaging submodule (22) and a gene identification submodule (23), wherein the PCR amplification submodule (21) is used to amplify gene deletion fragments, the gel imaging submodule (22) is used to observe the electrophoresis results of PCR products and screen target strains, and the gene identification submodule (23) is used to perform gene sequencing on the screened target strains.

5. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 1, characterized in that: The outer membrane vesicle production promotion module (3) includes an artificial induction submodule (31), an induction environment creation submodule (32) and an induction time control submodule (33), and the induction environment creation submodule (32) is connected to the artificial induction submodule (31) and the induction time control submodule (33) respectively. The artificial induction submodule (31) is used to set the induction conditions, the induction environment creation submodule (32) is used to execute the conditions set by the artificial induction submodule (31) to create an induction environment, and the induction time control submodule (33) is used to control the induction time.

6. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 5, characterized in that: The artificial inducer module (31) includes a chemical induction unit (311) and a physical induction unit (312). The chemical induction unit (311) is used to set chemical induction conditions, including the composition and dosage of the inducer, and the physical induction unit (312) is used to set physical induction conditions.

7. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 1, characterized in that: The outer membrane vesicle extraction module (4) includes an ultrasonic crushing treatment submodule (41) and a differential centrifugation treatment submodule (42), and the ultrasonic crushing treatment submodule (41) is connected to the differential centrifugation treatment submodule (42), the ultrasonic crushing treatment submodule (41) is used for ultrasonically treating Brucella, and the differential centrifugation treatment submodule (42) is used for extracting outer membrane vesicles.

8. The system for preparing outer membrane vesicles with guaranteed immune effect according to claim 1, characterized in that: The immune efficacy evaluation module (6) includes a cell level evaluation submodule (61) and an animal level evaluation submodule (62). The cell level evaluation submodule (61) is used to evaluate the immune efficacy of outer membrane vesicles at the cell level, and the animal level evaluation submodule (62) is used to evaluate the immune efficacy of outer membrane vesicles at the animal level.

9. A method for preparing outer membrane vesicles with guaranteed immune effects, comprising step 1, obtaining a strain; step 2, culturing the strain; step 3, promoting outer membrane vesicle secretion; step 4, extracting and purifying the outer membrane vesicles; and step 5, evaluating immune efficacy and yield; characterized in that: In the above step 1, a recombinant vector construction submodule (11) is used to perform a gene modification operation, and then a recombinant vector is constructed. The recombinant vector is then introduced into a competent cell of Brucella using a recombinant vector introduction submodule (12), and the gene on the chromosome is replaced by homologous recombination to obtain a genetically modified Brucella; In the above step 2, the Brucella obtained in step 1 is cultured using the strain culture submodule (13), and then the Brucella with successful genetic modification is screened using the strain screening module (2), and cultured to obtain the target bacteria; In the above step 3, the outer membrane vesicle production promotion module (3) is used to induce the target bacteria in step 2 to secrete outer membrane vesicles; In the above step 4, the target bacteria after the induction treatment in step 3 are collected, the outer membrane vesicles are extracted using the outer membrane vesicle extraction module (4), and the extracted outer membrane vesicles are purified using the outer membrane vesicle purification module (5); In the above step 5, the outer membrane vesicles purified in step 4 are collected, and the immune efficacy of the outer membrane vesicles is evaluated using the immune efficacy evaluation module (6), and the yield evaluation module (7) is used to evaluate the yield of the outer membrane vesicles.

10. The method for preparing outer membrane vesicles with guaranteed immune effect according to claim 9, characterized in that: In the step 1, in the gene modification operation, the gene to be knocked out is the alr gene.

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