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Bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins as well as construction method and application of bacterial outer membrane vesicle

A technology of outer membrane vesicles and construction methods, which is applied in the fields of synthetic biology and immunology, can solve problems that cannot be achieved, cannot be satisfied, and cannot be simultaneously loaded with multiple heterologous peptides or proteins, and achieve the effect of broadening the technical basis

Pending Publication Date: 2022-06-28
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the periplasmic space of prokaryotes provides the enzymes and oxidative conditions required for protein folding and disulfide bond formation, this greatly broadens the types of heterologous peptides or proteins that can be loaded in the inner cavity of outer membrane vesicles. The cavity position can protect heterologous peptides or proteins from degradation by external factors, but the method of loading heterologous peptides or proteins in the cavity cannot realize some functions of displaying heterologous peptides or proteins on the membrane surface, such as targeting function
[0005] Although the surface or lumen of the membrane can realize the display or loading of heterologous peptides or proteins on the outer membrane vesicles, the current existing technology cannot realize the simultaneous loading of multiple heterologous peptides or proteins on the outer membrane vesicles, thus failing to meet the requirements of the outer membrane vesicles. The need for vesicles as multivalent antigen delivery vehicles and multifunctional disease-targeted therapeutic vehicles

Method used

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  • Bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins as well as construction method and application of bacterial outer membrane vesicle
  • Bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins as well as construction method and application of bacterial outer membrane vesicle
  • Bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins as well as construction method and application of bacterial outer membrane vesicle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0042] Example 1: The preferred method for constructing an engineered strain capable of carrying multiple heterologous peptides or proteins with outer membrane vesicles in high yield

[0043] 1. Preparation of EcN-pKD46 electrocompetent cells with E. coli Nissle 1917 as the original strain:

[0044] 1.1 Transfer the pKD46 plasmid into EcN competent cells to obtain EcN-pKD46 (culture temperature: 30°C);

[0045] 1.2 Inoculate EcN-pKD46 into 50ml of LB medium, culture at 30°C and 220rpm until OD=0.15, add arabinose inducer with a final concentration of 30mm to induce the expression of the three enzymes of pKD46 plasmid, and continue to culture until OD=0.4 ;

[0046] 1.3 Take out the bacterial culture bottle and take an ice bath for 30min, then collect the bacteria by centrifugation at 4°C and 3500rpm;

[0047] 1.4 Resuspend with pre-cooled sterile water, centrifuge, remove the supernatant, repeat the washing once, and then centrifuge again;

[0048] 1.5 Resuspend with pre-co...

Embodiment example 2

[0081] Implementation case 2: Extraction method of NR-OMV

[0082] 1. The engineered bacteria EcN-NRΔnIpl was streaked on the LB medium with double resistance to chloramphenicol (25ng / μl) and ampicillin (100ng / μl), and cultured in a 37°C incubator for 12h;

[0083] 2. The next day, pick a single clone and inoculate it in 5ml LB culture at 37°C, 220rpm for 12h;

[0084] 3. Inoculate in 200ml LB medium at 1% and culture to OD=0.5, add arabinose to a final concentration of 30mm, and then culture at 37°C and 220rpm for 18h.

[0085] 4. Collect the bacterial culture supernatant in a pre-cooled centrifuge at 4°C at 12,000 rpm, filter the supernatant with a 0.45 μm filter, and then concentrate it with a 100 kDa ultrafiltration tube. After concentrating to 10 ml, use the outer membrane vesicles to extract The kit was extracted, and the protein concentration was measured by BCA method, and the samples were stored at -80°C.

[0086] 5. Take an appropriate amount of modified OMV sample...

Embodiment example 3

[0089] Example 3: Application of outer membrane vesicles loaded with multiple heterologous peptides or proteins as novel coronavirus antigen delivery carriers

[0090] 1. Dendritic cells (DC2.4) take up outer membrane vesicles (NR-OMV):

[0091] 1.1 WT-OMV and NR-OMV were incubated with DiD dye (1:500) for 30 min respectively, and the unbound DiD dye was washed away with a 100kDa ultrafiltration tube;

[0092] 1.2 Press 3×10 4 The cell density of each well DC2.4 cells were seeded in 24-well plates. After culturing adherent cells, WT-OMV (DiD) and NR-OMV (DiD) were added and incubated for 4 h. DC2.4 cells were collected, centrifuged, and washed with PBS. Cells were detected three times by flow cytometry;

[0093] 2. Expression of outer membrane vesicles (NR-OMV) stimulated dendritic cells (BMDC)

[0094] The primary bone marrow cells of BALB / c mice were taken and induced to differentiate into immature BMDC cells with GM-CSF (20ng / ml) and IL-4 (10ng / ml) in vitro; PBS, RBD, WT-O...

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Abstract

The invention discloses a bacterial outer membrane vesicle capable of presenting a plurality of heterologous peptides or proteins as well as a construction method and application of the bacterial outer membrane vesicle. A plurality of heterologous peptides or proteins are simultaneously loaded on the membrane surface of the outer membrane vesicle and the inner cavity of the vesicle, and the heterologous peptides or proteins anchored on the surface of the outer membrane vesicle are expressed and displayed on the surface of the outer membrane vesicle through linker fusion bacterial membrane protein. The heterologous peptide or protein loaded in the inner cavity of the outer membrane vesicle is expressed by fusing protein signal peptide or protein truncation of a cross-bacterial inner membrane so as to be loaded in the inner cavity of the outer membrane vesicle, and related gene knockout is carried out on gram-negative bacteria, so that efficient production of the outer membrane vesicle capable of presenting the heterologous peptide or protein is realized. According to the invention, the limitation of the quantity and the variety of the heterologous peptides or proteins loaded on the outer membrane vesicles and the application range of the outer membrane vesicles as a multi-antigen vaccine carrier are expanded, and the recombinant gram-negative bacteria can efficiently produce the outer membrane vesicles loaded with a plurality of heterologous peptides or proteins.

Description

technical field [0001] The invention belongs to the technical fields of synthetic biology and immunology, and particularly relates to a bacterial outer membrane vesicle capable of presenting multiple heterologous peptides or proteins, and a construction method and application thereof. Background technique [0002] Outer Membrane Vesicle (OMV) is a natural non-replicating vesicle-like structure with a size between 40-200 nm produced by blebbing from the outer membrane of Gram-negative bacteria, and its composition is rich in extracellular Membrane proteins, phospholipids and lipopolysaccharides, the inner cavity contains nucleic acid substances, enzymes, virulence factors and other molecules derived from parental strains, outer membrane vesicles play an important role in the exchange of information between bacteria and bacteria or between bacteria and hosts Role. [0003] Two natural advantages of outer membrane vesicles make them have excellent immune activation ability, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N1/21A61K39/385A61K39/39A61K39/215A61P31/14C12R1/19
CPCC12N15/70C07K14/005C12N15/625A61K39/385A61K39/39A61K39/12A61P31/14C12N2770/20022C12N2770/20034C07K2319/035C07K2319/02A61K2039/55594A61K2039/6006
Inventor 叶邦策吕召勇沃静
Owner ZHEJIANG UNIV OF TECH
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