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Prokaryotic exosome and extraction and separation method and application thereof

A prokaryotes, separation method technology, applied in the direction of separation of microorganisms, bacteria, etc., can solve problems such as unreported

Pending Publication Date: 2020-03-17
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are very few reports on the extraction of exosomes from prokaryotes, and the extraction of exosomes from lactic acid bacteria has not been reported.

Method used

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  • Prokaryotic exosome and extraction and separation method and application thereof
  • Prokaryotic exosome and extraction and separation method and application thereof
  • Prokaryotic exosome and extraction and separation method and application thereof

Examples

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Effect test

Embodiment 1

[0028] A method for extracting and separating prokaryotic exosomes. In this embodiment, prokaryotic organisms are lactic acid bacteria. See figure 1 , the specific steps of the method are as follows:

[0029] Step 1. Put the lactic acid bacteria into the MRS medium and incubate anaerobically at 37°C for 18 hours, then inoculate the second-generation bacteria solution that has been activated for 18 hours into 500 mL of MRS liquid medium according to the inoculum amount of 1 vt.%, and incubate at 37°C , the absorbance OD value of the culture solution at 600 nm is 2.2 and the minimum separation volume is not less than 200 mL, put it into a centrifuge at 4 °C, 5000 × g for 5 min, and take the supernatant of the lactic acid bacteria culture medium.

[0030] Step 2. Then centrifuge the supernatant of the lactic acid bacteria culture medium at 4°C, 2000×g for 10 min to take the supernatant of the second centrifugation liquid, transfer the supernatant to a new 50 mL centrifuge tube, a...

Embodiment 2

[0037] The lactic acid bacteria exosomes extracted and separated in Example 1 are subjected to nanoparticle tracking detection, and the nanoparticle tracking detection instrument is the ZetaVIEW S / N 17-310 nanometer particle size particle tracking analyzer produced by PARTICLE METRIX Company, and the software version used for analysis for ZetaView 8.04.02. The specific detection method is as follows: take a frozen sample at -80°C, thaw it in a 25°C water bath, and put it on ice immediately after thawing; then add 1×PBS for dilution, and directly use it for nanoparticle tracking detection. For the particle size detection and analysis chart of lactic acid bacteria exosomes, see figure 2 , it can be seen from the figure that the particle size of lactic acid bacteria exosomes detected by the nanoparticle size particle tracking analyzer meets the reported particle size detection standards of exosomes.

[0038] The exosomes extracted and separated in Example 1 were subjected to na...

Embodiment 3

[0040] The method described in Example 1 is applied in the extraction and separation of exosomes from prokaryotic organisms other than lactic acid bacteria.

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Abstract

The invention discloses a prokaryotic exosome and an extraction and separation method and application thereof, which belongs to the technical field of exosome extraction methods. The method comprisesthe steps of carrying out fermentation culture on prokaryotes, and centrifuging to acquire fermentation liquor; and carrying out multiple times of ultracentrifugation on the fermentation liquor to finally acquire the prokaryote exosome. The characteristics of the prokaryote exosome are identified through particle size analysis and nano transmission electron microscope analysis. According to the invention, the particle size of the prokaryotic exosome extracted and separated by the method integrally meets the standard, and the particle concentration can reach E + 9 or above; a clear vesicular structure can be seen in the field of view of an electron microscope, and the particle size of vesicles meets the detection standard of the exosome; the prokaryotic exosome extracted and separated by the method can be applied to transcriptomics, proteomics and lipidomics correlation analysis and preparation of an exosome drug loading system.

Description

technical field [0001] The invention belongs to the technical field of exosome extraction methods, and in particular relates to a prokaryotic exosome and its extraction and separation method and application. Background technique [0002] Lactic acid bacteria (LAB) is a general term for a group of bacteria that can use fermentable carbohydrates to produce large amounts of lactic acid. Such bacteria are widely distributed in nature and usually exist in foods such as meat, milk and vegetables and their products. In addition, lactic acid bacteria are also widely present in the intestines of livestock and poultry and a few clinical samples. Among them, lactic acid bacteria in the oral cavity and intestinal tract of humans and other mammals are important members of the normal microbial flora in a specific area. Rich species diversity, including at least 18 genera, a total of more than 200 species. One of the main functions of lactic acid bacteria includes improving the intesti...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02
CPCC12N1/20C12N1/02
Inventor 张嘉鑫陈莹艳徐珏郭宇星潘道东
Owner NANJING NORMAL UNIVERSITY
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