Bacterial outer membrane vesicle, universal nano vaccine containing bacterial outer membrane vesicle as well as preparation method and application thereof

A technology of outer membrane vesicles and nano-vaccine, which is applied in the direction of medical preparations containing active ingredients, nanotechnology, nanotechnology, etc., can solve the problems that do not involve tumor antigens and specific immunity, and achieve good tumor suppression effect, biological Good compatibility, the effect of inhibiting lung metastasis

Active Publication Date: 2021-03-12
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are still few studies on the use of OMV’s immunostimulatory function in tumor immunotherapy. At present, there is only one report from a Korean research group, using the natural immune response stimulated by Escherichia coli-derived OMV to significantly inhibit tumor growth, but this One study did not address tumor antigens and specific immune

Method used

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  • Bacterial outer membrane vesicle, universal nano vaccine containing bacterial outer membrane vesicle as well as preparation method and application thereof
  • Bacterial outer membrane vesicle, universal nano vaccine containing bacterial outer membrane vesicle as well as preparation method and application thereof
  • Bacterial outer membrane vesicle, universal nano vaccine containing bacterial outer membrane vesicle as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] In this embodiment, the OMV nano-vaccine platform and tag antigen library are constructed by the following methods, which are:

[0054] Select Mark Howarth et al., Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(12): 4347-4348 and Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(5): 1202 The molecular glue SpyCatcher, SpyTag, SnoopCatcher, and SnoopTag provided by -1207 are fused to express SpyCatcher and SnoopCatcher with the C-terminus of ClyA protein through genetic engineering, and the plasmid pETDuet-ClyA-SpyCatcher-ClyA-SnoopCatcher is constructed, and then the plasmid is transformed Enter Rosetta (DE3), and express the target protein under the action of 1mM IPTG inducer. The OMV was isolated and purified by ultrafiltration and ultracentrifugation. Among them, SpyCatcher can be specific to SpyTag (valine-proline-threonine-isoleucine-valine-methionine-valine-aspartic acid-alanine-...

Embodiment 2

[0070] The purpose of this example is to verify that the tag and the OMV vaccine platform can be stably connected.

[0071] In order to detect the binding of the label to the corresponding molecular glue protein, this embodiment couples a section of HA polypeptide (YPYDVPDYA) to SpyTag and SnoopTag respectively, and the HA label can be detected by Western blotting. The corresponding molecular glue SpyCatcher and SnoopCatcher proteins can be detected at a position of about 45kDa. In this embodiment, it is judged whether the reaction between the Catcher and the Tag occurs by detecting the position of the HA tag. Add 0, 10, 20, 30, and 40 μg ClyA-CatchersOMV to 50 μg SpyTag-HA or SnoopTag-HA, respectively, and react at room temperature for half an hour, then add protein loading buffer to denature the protein (100 denaturation for 10 min). By SDS-PAGE electrophoresis, the HA-labeled antibody was incubated after transfer to the membrane, and the position of the band was detected b...

Embodiment 3

[0074] The purpose of this example is to determine that the nano-vaccine platform and its drug combination can effectively stimulate DC maturation and realize antigen presentation, which can be used as a potential stimulator of DC vaccine.

[0075] In this example, a tagged antigen with OVA antigen peptide, ie SpyTag-OVA, was synthesized by solid phase synthesis. The nano vaccine preparation (CC-SpyTag-OVAOMV) was prepared after mixing with ClyA-Cathcers OMV. In vitro, this example extracts mouse thigh bone marrow-derived cells, induces bone marrow-derived cells to differentiate into dendritic cells (DC cells) through co-stimulation with GM-CSF and IL-4, and adds CC-SpT-OVA OMV and other controls The group stimulated DC cells, and detected the maturation and antigen presentation of DC cells by flow cytometry and confocal laser. Such as image 3 As shown, the addition of OMV can effectively stimulate DC cell maturation, and DC cell maturation markers were significantly up-reg...

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Abstract

The invention relates to a bacterial outer membrane vesicle, a nano vaccine containing the bacterial outer membrane vesicle as well as a preparation method and application thereof. The bacterial outermembrane vesicle contains molecular gluewater protein SpyCatcher and/or SnoopCatcher, the tumor vaccine comprises the bacterial outer membrane vesicle and an antigen connected with the bacterial outer membrane vesicle in the form of an isopeptide bond, and the antigen carries SpyTag and/or SnoopTag. According to the invention, the bacterial outer membrane vesicle containing molecular gluewater protein SpyCatcher and SnoopCatcher is used as a tumor vaccine platform and is connected with the antigen carrying SpyTag and/or SnoopTag to obtain the tumor vaccine, and the tumor vaccine can effectively activate an organism to generate innate immunity and realize specific killing of a plurality of antigens at the same time. According to the method, personalized tumor vaccines for tumors from different sources can be rapidly obtained.

Description

technical field [0001] The invention relates to the technical field of nano-medicines, in particular to a bacterial outer membrane vesicle, a universal nano-vaccine containing the bacterial outer membrane vesicle, a preparation method and application thereof. Background technique [0002] In recent years, with the development of molecular biology and tumor immunology, tumor immunotherapy has become the most potential tumor treatment after surgery, chemotherapy, radiotherapy and molecular targeted therapy. Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the immune system's recognition and monitoring of tumors, and restores the body's normal anti-tumor immune response. It mainly includes immune checkpoint inhibitors, tumor vaccines, and cell therapy. and cytokine therapy. In recent years, tumor immunotherapy has developed rapidly, and it has demonstrated strong anti-tumor activity in the treatment of various tumors s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/385A61K39/00A61P35/00B82Y5/00B82Y40/00
CPCA61K39/39A61K39/385A61K39/0011A61K39/00119A61P35/00B82Y5/00B82Y40/00A61K2039/55594A61K2039/55516A61K2039/5158Y02A50/30
Inventor 聂广军赵潇程科满赵宇亮
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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