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WO2025200475A1 - Système et procédé de préparation de vésicules à membrane externe à effets immunitaires garantis - Google Patents

Système et procédé de préparation de vésicules à membrane externe à effets immunitaires garantis

Info

Publication number
WO2025200475A1
WO2025200475A1 PCT/CN2024/131238 CN2024131238W WO2025200475A1 WO 2025200475 A1 WO2025200475 A1 WO 2025200475A1 CN 2024131238 W CN2024131238 W CN 2024131238W WO 2025200475 A1 WO2025200475 A1 WO 2025200475A1
Authority
WO
WIPO (PCT)
Prior art keywords
outer membrane
submodule
module
culture
membrane vesicles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/131238
Other languages
English (en)
Chinese (zh)
Inventor
郝明月
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hubei University of Medicine
Original Assignee
Hubei University of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hubei University of Medicine filed Critical Hubei University of Medicine
Priority to PCT/CN2024/131238 priority Critical patent/WO2025200475A1/fr
Publication of WO2025200475A1 publication Critical patent/WO2025200475A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/098—Brucella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 introduce the recombinant vector into the competent cells of Brucella
  • the strain culture submodule is used to culture Brucella
  • the culture condition control submodule is used to control the culture conditions of Brucella.
  • 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
  • the gene identification submodule is used to perform gene sequencing on the screened target strains.
  • 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.
  • 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
  • the animal level evaluation submodule is used to evaluate the immune efficacy of outer membrane vesicles at the animal level.
  • 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;
  • the outer membrane vesicle production promotion module is used to induce the target bacteria in step 2 to secrete outer membrane vesicles;
  • 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;
  • 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.
  • the gene knocked out is the alr gene.
  • FIG1 is a block diagram of the system structure of the present invention.
  • FIG2 is a block diagram of the bacterial strain acquisition module of the present invention.
  • FIG3 is a structural block diagram of an outer membrane vesicle production promotion module of the present invention.
  • FIG5 is a flow chart of the method of the present invention.
  • 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.
  • the outer membrane vesicle preparation system includes 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, and 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 1 is used to obtain the target strain, and the strain screening module 2 is used to screen the strains that have been successfully genetically modified.
  • 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;
  • the gene knocked out is alr gene;
  • the outer membrane vesicle production promotion module 3 is used to induce the purpose of step 2
  • 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;
  • 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.
  • 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 successfully genetically modified, and it is cultivated to obtain the target thalline, and in the culture process, the culture condition control submodule 14 is utilized to control the culture environment in the optimum condition, specifically: the culture medium component control unit 141 is utilized to control the nutrients and ion concentration of the culture medium, the culture temperature control unit 142
  • the 143 controls the oxygen concentration of the culture environment, uses the pH control unit 144 to control the pH of the culture environment, and uses the culture time control unit 145 to control the culture time; uses the strain screening module 2 to screen specifically: uses the PCR amplification submodule 21 to perform PCR amplification, uses the gel imaging submodule 22 to observe the electrophoresis results of the PCR products, screens out the Brucella with successful gene modification, and then uses the gene identification submodule 23 to perform gene sequencing to further identify whether the gene modification is successful; uses the outer membrane vesicle production promotion module 3 to induce the target bacteria to secrete outer membrane vesicles, specifically: uses the chemical induction unit 311 to set chemical induction conditions, including the composition and dosage of the inducer, uses the physical induction unit 312 to set physical induction conditions, uses the induction environment creation submodule 32 to execute the conditions set by the artificial induction submodule 31, creates an induction environment, thereby promoting the target bacteria to secrete a large amount of outer membrane vesicles

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un système et un procédé de préparation de vésicules à membrane externe à effets immunitaires garantis. Le système comprend un module d'acquisition de souche, un module de criblage de souche, un module de promotion de rendement de vésicules à membrane externe, un module d'extraction de vésicules à membrane externe, un module de purification de vésicules à membrane externe, un module d'évaluation d'efficacité immunitaire et un module d'évaluation de rendement, le module d'acquisition de souche étant relié au module de criblage de souche, au module de promotion de rendement de vésicules à membrane externe et au module d'extraction de vésicules à membrane externe, respectivement. Au moyen d'une opération de modification génétique, le gène alr de Brucella est inactivé, ce qui à son tour améliore le rendement et l'efficacité immunitaire des vésicules à membrane externe. De plus, au moyen de la régulation stricte des conditions de culture de Brucella, un environnement d'induction est établi pour induire Brucella pour sécréter des vésicules à membrane externe, ce qui permet d'améliorer davantage le rendement de vésicules à membrane externe, et d'obtenir à son tour des vésicules à membrane externe avec un rendement élevé et de bons effets immunitaires, ce qui répond aux exigences de production de vaccin à grande échelle, et fournit un adjuvant de vaccin de haute qualité pour la recherche, le développement et l'application de vaccins à Brucella.
PCT/CN2024/131238 2024-11-11 2024-11-11 Système et procédé de préparation de vésicules à membrane externe à effets immunitaires garantis Pending WO2025200475A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/131238 WO2025200475A1 (fr) 2024-11-11 2024-11-11 Système et procédé de préparation de vésicules à membrane externe à effets immunitaires garantis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/131238 WO2025200475A1 (fr) 2024-11-11 2024-11-11 Système et procédé de préparation de vésicules à membrane externe à effets immunitaires garantis

Publications (1)

Publication Number Publication Date
WO2025200475A1 true WO2025200475A1 (fr) 2025-10-02

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ID=97219157

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/131238 Pending WO2025200475A1 (fr) 2024-11-11 2024-11-11 Système et procédé de préparation de vésicules à membrane externe à effets immunitaires garantis

Country Status (1)

Country Link
WO (1) WO2025200475A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200023051A1 (en) * 2016-06-29 2020-01-23 Glaxosmithkline Biologicals, S.A. Immunogenic compositions
CN111433348A (zh) * 2017-12-04 2020-07-17 由卫生福利和体育大臣代表的荷兰王国 一种改良的生产外膜囊泡的方法
CN113025640A (zh) * 2021-03-17 2021-06-25 天康生物制药有限公司 一种布鲁氏菌外膜囊泡的制备方法及其应用
CN114921398A (zh) * 2022-06-17 2022-08-19 南昌大学 一种制备沙门菌外膜囊泡的方法及其应用
CN116096871A (zh) * 2020-09-11 2023-05-09 葛兰素史克生物有限公司 外膜囊泡

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200023051A1 (en) * 2016-06-29 2020-01-23 Glaxosmithkline Biologicals, S.A. Immunogenic compositions
CN111433348A (zh) * 2017-12-04 2020-07-17 由卫生福利和体育大臣代表的荷兰王国 一种改良的生产外膜囊泡的方法
CN116096871A (zh) * 2020-09-11 2023-05-09 葛兰素史克生物有限公司 外膜囊泡
CN113025640A (zh) * 2021-03-17 2021-06-25 天康生物制药有限公司 一种布鲁氏菌外膜囊泡的制备方法及其应用
CN114921398A (zh) * 2022-06-17 2022-08-19 南昌大学 一种制备沙门菌外膜囊泡的方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAO, MINGYUE ET AL.: "Deletion of the Alr Gene in Brucella suis S2 Attenuates Virulence by Enhancing TLR4-NF-κB-NLRP3-mediated Host Inflammatory Responses", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 137, 18 June 2024 (2024-06-18), XP087558452, DOI: 10.1016/j.intimp.2024.112443 *

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