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Method for carrying out bio-orthogonal polysaccharide labeling from microbiome level, and applications thereof

A bio-orthogonal and microbial technology, applied in the field of chemical biology, can solve the problems that bacteria cannot be studied, the operation is cumbersome, and they are helpless

Inactive Publication Date: 2018-03-06
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the reports on bio-orthogonal glycan labeling in bacteria are limited to screening for a specific bacteria that can be cultured alone, the efficiency is low, and for bacteria that cannot be cultured alone (generally considered to account for more than 95% of all bacteria) ) helpless
The same problem exists in the discovery of new glycoproteins in bacteria. It is often impossible to study bacteria that cannot be cultured alone, and it is impossible to understand the potential glycoproteins.
[0006] The above-mentioned commonly used methods for microbiome imaging have the following problems (1) The disadvantage of the method of inserting fluorescent proteins is that >80% of intestinal commensal microorganisms cannot be cultured in vitro alone, and thus cannot be transgenic
(2) FISH has problems such as complex DNA probe design, low rRNA content, cumbersome operation, etc., and is limited to the detection of bacteria whose existence is known in advance and whose primer feasibility has been verified, so its application is still subject to many restrictions
(3) The method of using chemical biology to label intestinal commensal bacteria has the disadvantage that specific bacteria still need to be cultured separately

Method used

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  • Method for carrying out bio-orthogonal polysaccharide labeling from microbiome level, and applications thereof
  • Method for carrying out bio-orthogonal polysaccharide labeling from microbiome level, and applications thereof
  • Method for carrying out bio-orthogonal polysaccharide labeling from microbiome level, and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0043] Three mice from clean-grade (specific-pathogen-free, SPF) C57 / BL6 adult mice (8-12 weeks) were divided into three groups, with one mouse in each group. After the large intestine and small intestine were taken through aseptic operation, they were quickly ground with a tissue grinder and filtered with a sieve (70mesh). The filtrate was diluted and evenly spread on a special intestinal microbiome medium (modifiedGifu anaerobe media ) (see Table 1 for the culture media corresponding to the three groups), 1 mM non-natural sugar probes have been added to the culture media of the experimental group in advance. Bacteria were collected after culturing in an anaerobic environment at 37°C for 5-7 days, and a fluorescent group was coupled to a cycloaddition reaction of azide and alkyne catalyzed by copper.

[0044] Table 1:

[0045]

[0046]

[0047] Afterwards, the samples were observed under a fluorescence microscope, and the observation results were as follows: Figure 1a...

Embodiment 2

[0052] Adopt the method identical with embodiment 1, use Ac 4 GalNAz or Ac 4 GlcNAz metabolically labeled bacterial populations (about 20mg), washed twice with PBS, lysed (sonicated or using bacterial lysate), centrifuged (10,000x g, 15min), supernatant was taken, and alkynyl biotin (0.1 mM), using copper to catalyze the cycloaddition reaction of azide and alkyne, after coupling the azide-labeled bacterial glycoprotein to biotin, the protein was precipitated with anhydrous methanol, centrifuged (4,000x g, 15min), and then used After washing with methanol for three times, use protein reconstitution solution to redissolve the protein, add strepavidin agarose microspheres to enrich the biotin-labeled protein, wash the microspheres thoroughly to remove non-specifically adsorbed protein, and finally use the loading buffer used for SDS-PAGE to load the protein The enriched glycoproteins are detached from the microspheres, and the proteins in the samples are analyzed by SDS-PAGE, an...

Embodiment 3

[0056] Use 8AzKDO to metabolically label the complete flora (20mg), in which Gram-negative bacteria are specifically labeled, and use PBS after coupling alkynyl-TAMRA (20μM) through the cycloaddition reaction of azide and alkyne catalyzed by copper. After washing 5 times, the finally obtained bacteria were resuspended in 500 μL PBS. Model mice (C57 / BL6) were intragastrically administered (200 μL) using this sample, and 4 to 6 hours later, the distribution of Gram-negative bacteria in the mouse intestine was observed. Observation methods include using two-photon microscopy to perform in vivo imaging of the flora in the intestinal tract of mice, or to isolate the intestinal tract of mice and perform frozen section processing, and use fluorescence microscopy to detect the presence of Gram-negative bacteria in the intestinal tract distribution is observed.

[0057] After the intestinal microbiome metabolically labeled with 8AzKDO probe (1mM) was administered to the mice, the smal...

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Abstract

The present invention provides a method for carrying out bio-orthogonal polysaccharide labeling from a microbiome level. The method comprises: placing microbial flora in a culture medium added with afirst probe, and culturing to obtain a microbial flora labeled with the first probe, wherein the first probe is one or a variety of unnatural saccharides; and carrying out a bio-orthogonal reaction onthe microbial flora labeled with the first probe and a second probe to obtain a microbial flora labeled with the second probe, wherein the second probe is a chemical marker capable of being subjectedto a bio-orthogonal reaction with the first probe. According to the present invention, with the method, a variety of bacteria capable of being metabolized and labeled with unnatural saccharides can be rapidly screened in the high-throughput manner, the specific types of the unnatural saccharides for metabolizing and labeling different bacteria can be determined, the possibility can be provided for the in vivo imaging of the specific microbiome, and the powerful tool is provided for the exploring of the biological functions of the macromolecules containing the polysaccharide structure in the bacteria difficultly cultured separately.

Description

technical field [0001] The invention relates to the field of chemical biology, in particular to a method and application of bio-orthogonal glycan labeling at the microbiome level. Background technique [0002] Using chemical biology methods as an entry point, labeling cellular glycan structures by introducing precursor monosaccharide analogs (non-natural sugars) containing bioorthogonal groups such as alkyne or azide during the biosynthesis of glycans the goal of. Through the design of non-natural sugar probes, they can enter the sugar biosynthesis mechanism of cells, pass through cell metabolism, and finally be expressed on the glycan-containing structures on the cell surface. Finally, probe molecules, such as fluorescent probes and biotin probes, can be introduced under living cell conditions through bioorthogonal reaction. Bioorthogonal glycan labeling has also been extensively reported in bacteria. In several representative studies, different non-natural sugar probes c...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/10G01N33/68
CPCC12Q1/04C12Q1/10G01N33/68
Inventor 陈兴王炜
Owner PEKING UNIV
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