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Bacterial outer membrane vesicle carrier as well as preparation method and application thereof

A technology for outer membrane vesicles and bacteria, applied in the fields of biochemical equipment and methods, bacteria, chemical instruments and methods, etc., can solve the problem of reduced antigen uptake, and achieve the effect of promoting cross-presentation and inducing specific immune responses.

Active Publication Date: 2021-05-11
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

While DC cell maturation is necessary for successful antigen presentation, premature dendritic cell maturation leads to greatly reduced antigen uptake by OMVs

Method used

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  • Bacterial outer membrane vesicle carrier as well as preparation method and application thereof
  • Bacterial outer membrane vesicle carrier as well as preparation method and application thereof
  • Bacterial outer membrane vesicle carrier as well as preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0058] In a third aspect, the present invention provides the preparation method of the vaccine carrier and nano vaccine, comprising: 1) the coding gene of the SpyCatcher protein and the B domain SPAb of Staphylococcus aureus protein A through genetic recombination and the coding of the ClyA protein After gene recombination, the bacterial outer membrane vesicles are obtained by fusion expression in Gram-negative bacteria; 2) the antigen containing SpyTag and the antibody targeting DC are mixed with the bacterial outer membrane vesicles respectively.

[0059] Further, the mass ratio of the SpyTag-containing antigen, the DC-targeting antibody and the bacterial outer membrane vesicle vaccine carrier is 1:1.

[0060] Preferably, the Gram-negative bacteria is Escherichia coli.

[0061] As a preferred technical solution, the present invention provides a method for preparing a nano-vaccine, the method comprising the following steps:

[0062] (1) Using genetic engineering methods to d...

Embodiment 1

[0073] Example 1 Preparation of Bacterial Outer Membrane Vesicle Carrier

[0074] SpyCatcher and the B domain SPAb of Staphylococcus aureus protein A and the C-terminus of ClyA protein were fused and expressed by genetic engineering. After the plasmid was constructed, the plasmid was transformed into Escherichia coli Rosetta (DE3), and the effect of IPTG inducer express the target protein. The universal vaccine vector OMV-SpyC-SPAb was obtained by separation and purification by means of ultrafiltration and ultracentrifugation. Utilize the laser particle size analyzer and transmission electron microscope to obtain the OMV vaccine universal carrier characterization, such as figure 1 as shown, figure 1 A in A is the particle size of the general carrier of OMV vaccine, and the particle size is 27.9nm; figure 1 B in the figure is the electron microscope topography of the prepared OMV vaccine universal carrier. Wherein, the construction method of the recombinant plasmid pETDuet-...

Embodiment 2

[0079] The purpose of this example is to verify whether the B domain SPAb of Staphylococcus aureus protein A binds to the antibody, and whether the reaction between SpyCatcher and SpyTag occurs.

[0080] The B domain SPab of Staphylococcus aureus protein A fused and expressed on the OMV universal vaccine vector of Example 1 can specifically adsorb antibodies; SpyCatcher can specifically react with SpyTag to form a stable isopeptide bond.

[0081] In order to detect the combination of SPAb and antibody, horseradish peroxidase HPR-labeled rat IgG antibody (RatIgG-HPR) was incubated with the universal vaccine carrier OMV-SpyC-SPAb, detected by western blot, and detected by adding developer The position of the band, SPAb successfully adsorbed Rat IgG-HPR, and detected the protein band of the B domain of Staphylococcus aureus protein A fusion expressed by SPAb and ClyA protein at the position of about 45KDa, see image 3 A in

[0082] In order to detect the SpyCatcher / SpyTag react...

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Abstract

The invention provides a bacterial outer membrane vesicle carrier as well as a preparation method and application thereof. The invention provides a vaccine universal vector OMV-SpyC-SPAb which is constructed on the basis of a B structural domain SPAb of staphylococcus aureus protein A, is based on bacterial outer membrane vesicles and can target dendritic cells, the B structural domain SPAb of the staphylococcus aureus protein A is displayed on the bacterial outer membrane vesicles and can be rapidly combined with an antibody (such as an Anti-DEC205 antibody) of the targeted dendritic cells, and a novel OMV-OVA-DEC vaccine capable of targeting the dendritic cells and obviously enhancing antigen intake is prepared by using the SpyCatcher / SpyTag molecular glue to display the antigen on the surface of the bacterial outer membrane vesicles, so that the cross presentation of the dendritic cells on the antigen can be promoted, and the specific immune response can be induced, thereby achieving the anti-tumor purpose and having a wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bacterial outer membrane vesicle carrier and its preparation method and application. Background technique [0002] As a kind of tumor immunotherapy, tumor vaccine has a history of more than one hundred years, and has shown potential advantages and good application prospects in tumor immunotherapy. However, the immunogenicity of single antigen therapy is poor, which often leads to unsatisfactory anti-tumor immune effect. In order to solve this problem, the use of vaccine carriers can efficiently deliver antigens and adjuvants, and then enhance the antigen-specific adaptive immune response by improving the uptake, processing and presentation efficiency of antigens by dendritic cells (DC) . At present, liposomes, nanoparticles, nanomicelles, hydrogels, and biologically derived materials based on biological materials are used as vaccine carriers, which can improve the delivery effici...

Claims

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Application Information

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IPC IPC(8): A61K47/46A61K9/127A61K47/68A61K47/69A61K39/00A61P35/00A61P35/04C12N15/70C12N15/62C12N1/21C12R1/19
CPCA61K47/46A61K9/127A61K47/68A61K47/6915A61K39/0011A61P35/00A61P35/04C12N15/70C07K14/31C07K2319/035C07K2319/735
Inventor 聂广军赵潇赵宇亮梁洁
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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