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Extracellular vesicles derived from gram-positive bacteria and uses thereof

A cell and application technology, applied in the field of extracellular vesicles, which can solve the problems of not disclosing relevant information and having no outer membrane

Active Publication Date: 2014-10-29
AEON MEDIX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for Gram-positive bacteria, which do not have an outer cell membrane, and the cell membrane is wrapped by the cell wall, there has been no disclosure so far of secreting extracellular vesicles or causing disease by extracellular vesicles derived from Gram-positive bacteria related information

Method used

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  • Extracellular vesicles derived from gram-positive bacteria and uses thereof
  • Extracellular vesicles derived from gram-positive bacteria and uses thereof
  • Extracellular vesicles derived from gram-positive bacteria and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0152] Example 1. Observation of Staphylococcus aureus cells by electron microscopy

[0153] Staphylococcus aureus (ATCC14458) was cultured in nutrient broth until the absorbance (600 nm) value reached 1.0, and the culture broth was centrifuged at 10,000 x g for 20 minutes. The precipitated Staphylococcus aureus cells were fixed (fix) with 2.5% glutaraldehyde (glutaraldehyde) for 2 hours, and then post-fixed (post-fix) with 1% osmium tetroxide (osmium tetroxide) for 1 hour, and then, with ethanol (ethanol) was dehydrated in stages to make epoxy resin modules, and ultrathin sections were made with a thickness of 70 nm. Cell slices were placed on a glow-discharged carbon-coated copper grid for 3 minutes and then stained with 2% uranyl acetate and lead calcium citrate ( staining), and observed by JEM101 (Jeol, Japan) transmission electron microscope (transmission electron microscope, TEM). Such as figure 1 As shown in the transmission electron microscope image of the Staphyloc...

Embodiment 2

[0156] Example 2. Preparation of extracellular vesicles derived from Staphylococcus aureus

[0157] [Common Extracellular Vesicle Isolation Method]

[0158] Staphylococcus aureus was inoculated in a test tube containing 3 ml of nutrient solution at 37 o C for 6 hours, transfer 5 ml of it into a 2 L Erlenmeyer flask filled with 500 ml of nutrient solution, at 37 o Incubate for 4 hours under C condition to make the absorbance (600 nm) value reach 1.0. Put the culture solution into a 500 ml capacity high speed centrifuge tube (high speed centrifuge tube), in 4 o Under C conditions, centrifuge at 10,000 x g for 20 minutes. The supernatant from which bacteria were removed was filtered once through a membrane filter (membrane filter) with a pore size of 0.45 μm, and then concentrated 25 times using an ultrafiltration system (Quixstand system) equipped with a membrane capable of removing proteins with a molecular weight of less than 100 kDa , the concentrated solution was filtere...

Embodiment 3

[0161] Example 3. Characterization of extracellular vesicles derived from Staphylococcus aureus

[0162] The extracellular vesicles isolated from Staphylococcus aureus according to the method described in Example 2 were placed on a glow discharge carbon-coated copper grid for adsorption for 3 minutes, the grid was rinsed with distilled water, and stained with 2% uranyl acetate. And observed by JEM101 transmission electron microscope.

[0163] Such as image 3 The transmission electron microscope image of a shows that the extracellular vesicles derived from S. aureus form closed spheres with a size of 20-100 nm. The isolated extracellular vesicles were pasted on a cover glass, fixed with 2.5% glutaraldehyde for 1 hour, post-fixed with 1% osmium tetroxide for 1 hour, dehydrated in stages with ethanol, and then , using CO 2 The system performs critical point drying. The cover glass with extracellular vesicles was placed on the sample stage and coated with platinum, and then ...

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Abstract

The present invention relates to extracellular vesicles (EVs) derived from gram-positive bacteria. In detail, the present invention provides animal models of disease using extracellular vesicles derived from gram-positive bacteria, provides a method for screening an active candidate substance which is capable of preventing or treating diseases through the animal models of disease, provides vaccines for preventing or treating diseases caused by extracellular vesicles derived from gram-positive bacteria, and provides a method for diagnosing the causative factors of diseases caused by gram-positive bacteria using extracellular vesicles.

Description

technical field [0001] The present invention relates to an extracellular vesicle (EV) derived from Gram-positive bacteria, a disease model using the vesicle, a method for screening candidate drugs, a vaccine, a method for diagnosing disease-causing factors, and the like. Background technique [0002] A gram-positive bacterium is characterized by being stained purple when gram-stained, and it is a bacterium without an outer membrane (outer membrane) compared to a gram-negative bacterium. From the point of view of taxonomy, Firmicutes, Actinomycetes, and Amutebacteria belong to Gram-positive bacteria. The characteristic of Firmicutes and Actinomycetes is that the peptidoglycan in the cell wall is very rich, and the amount of G+C in the genetic material of the former is relatively small. On the contrary, the amount of G+C in the latter is very large. Murphycota are characterized by the absence of cell walls. [0003] Most of the pathogenic bacteria that cause human diseases a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/00A61K9/133A61K35/74A61K39/00C12Q1/68G01N33/53C12R1/44
CPCA01K2267/0375C12P1/04A01K2207/00Y02P20/582A61P31/04A61P37/04A61P9/10C12Q1/6883C12Q2600/158
Inventor 高用松金润根李恩暎洪成旭金志贤崔昇振
Owner AEON MEDIX
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