CN-122011176-A - Method for microfluidic high-throughput screening of AKK bacteria

CN122011176ACN 122011176 ACN122011176 ACN 122011176ACN-122011176-A

Abstract

The invention discloses a microfluidic high-throughput screening method of AKK bacteria, which comprises the steps of 1) preparing a microfluidic chip, 2) collecting fresh feces of healthy volunteers, diluting, adding fluorescent labeled antibodies to obtain a disperse phase, 3) preparing liquid drops by taking paraffin oil and Span-80 as mobile phases, enabling the liquid drops to enter a liquid drop culture pond through an S-shaped liquid drop flow channel, culturing, introducing the mobile phases to enable the liquid drops in the liquid drop culture pond to be introduced into a liquid drop collecting pond, taking out the liquid drops, and culturing the liquid drops with fluorescent signals on an improved brain heart infusion broth solid culture medium through detecting fluorescent signals to obtain strains to be identified, 4) taking the strains to be identified as templates, carrying out PCR amplification, sequencing the amplification products, and comparing the sequencing results with BLAST sequences on NCBI websites.

Inventors

YI DEWEI
WU HAO
QIAO JIANJUN
QIAN YONGQING
REN SHUJIANG
LIU KE
XIONG HUI

Assignees

天津大学浙江研究院(绍兴)

Dates

Publication Date: 20260512
Application Date: 20260115

Claims (4)

1. A fluorescent-labeled antibody against AKK bacteria Amuc-1100 protein, characterized by being prepared by the steps of: Adding PBS buffer solution into an antibody aiming at AKK bacteria Amuc-1100 protein to prepare an antibody solution, adding fluorescein isothiocyanate into dimethyl sulfoxide to prepare fluorescein isothiocyanate-dimethyl sulfoxide solution, adding the fluorescein isothiocyanate-dimethyl sulfoxide solution into the antibody solution, shaking, mixing uniformly, incubating, eluting with reference to an antibody labeling kit instruction book, and purifying to obtain a fluorescence labeling antibody aiming at AKK bacteria Amuc-1100 protein, which is called as fluorescence labeling antibody for short; The antibody against AKK bacterium Amuc-1100 protein comprises a heavy chain variable region and a light chain variable region; HCDR1 in the heavy chain variable region 3 Has the amino acid sequence shown in SEQ ID NO.1 3 Is shown in the figure; LCDR1 in the light chain variable region 3 With the amino acid sequence shown in SEQ ID NO. 9 11.
2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is as shown in seq id No. 4.
3. The antibody of claim 1, wherein the amino acid sequence of the light chain variable region is as shown in seq id No. 12.
4. The microfluidic high-throughput screening method for AKK bacteria is characterized by comprising the following steps of: The method comprises the steps of 1) preparing a microfluidic chip, wherein the microfluidic chip comprises a square-shaped liquid drop generating channel (3), the middle part of the left channel of the square-shaped liquid drop generating channel (3) is connected with a mobile phase sample injection pool (1), the middle part of the right channel of the square-shaped liquid drop generating channel (3) is connected with a disperse phase sample injection pool (2), the middle part of the right channel of the square-shaped liquid drop generating channel (3) is connected with an S-shaped liquid drop circulating channel (4) through a channel and then is connected with a liquid drop culture pool (5) through a channel, and the liquid drop culture pool (5) is connected with a liquid drop collecting pool (6) through a channel; 2) Collecting fresh feces of healthy volunteers, performing 10 -3 -10 -5 gradient dilution in an anaerobic incubator by using sterilized normal saline to obtain a diluent, diluting the diluent by using a modified brain heart infusion broth culture medium, and adding the fluorescent-labeled antibody according to one of claims 1-3 to obtain a dispersed phase; 3) Under anaerobic culture conditions, taking mixed liquid of paraffin oil and Span-80 as a mobile phase, introducing the mobile phase into a mobile phase sample injection pool (1) of a microfluidic chip, introducing the dispersed phase into a dispersed phase sample injection pool (2) of the microfluidic chip, performing droplet preparation, introducing generated droplets into a droplet culture pool (5) through an S-shaped droplet flow channel (4), stopping introducing the dispersed phase and the mobile phase after the droplet culture pool (5) is filled with the droplets, culturing for 24-48 hours at the temperature of 35-40 ℃, re-introducing the mobile phase after culturing is finished, introducing the droplets in the droplet culture pool (5) into a droplet collecting pool (6), taking out, performing fluorescence microscopy detection, detecting fluorescent signals through a fluorescence microscopy receiver, and culturing the droplets with fluorescent signals on an improved brain heart immersion liquid solid broth culture medium for 24-48 hours to obtain a strain to be identified; 4) Taking a strain to be identified as a template, taking an upstream primer P1 and a downstream primer P2 as an upstream primer and a downstream primer, performing PCR (polymerase chain reaction) amplification, sequencing an amplification product, and performing BLAST sequence comparison on a NCBI (NCBI) website on the sequencing result to screen out a target strain AKK (alkyl ketene dimer) bacteria; The nucleotide sequence of the upstream primer P1 is shown in SEQ ID NO. 17; The nucleotide sequence of the downstream primer P2 is shown as SEQ ID NO. 18.

Description

Method for microfluidic high-throughput screening of AKK bacteria Technical Field The invention belongs to the technical field of microorganisms, and particularly relates to a microfluidic high-throughput screening method for AKK bacteria (AKKERMANSIA MUCINIPHILA, AKK). Background AKKERMANSIA MUCINIPHILA (A. Mucin Achromobacter, abbreviated as AKK) belongs to the phylum of the genus Microbacterium, is a gram-negative bacterium which is widely present in vertebrates and is mainly colonized in the mucous membrane mucus layers of the cecum and colon, accounting for 1% -3% of the total intestinal microorganisms. AKK bacteria grow in the gut and are able to utilize mucins secreted by the host as "food" to colonise the gut by competitive exclusion and protect the gut from pathogens. In recent years, research shows that AKK bacteria have beneficial effects on various pathologies, and the discovered medicinal effects are quite wide. Has been widely reported to have various effects related to anticancer, anti-aging, lipid-lowering and anti-inflammatory, immunity-enhancing, nervous system function-regulating and the like. Studies have shown that oral administration of 1 x 10 10 doses of AKK bacteria, both live and dead, to human volunteers is very safe and does not show adverse effects. This makes AKK bacteria one of the most potential next generation probiotics. Although AKK bacteria have a number of potential uses, it is very difficult to isolate and purify as strict anaerobes. Since AKK bacteria were first isolated from human feces by Derrien et al in 2004, medium-coated screening has been used as the dominant screening method. However, the coating method for screening AKK bacteria has long screening period and poor specificity, which brings challenges to rapid screening of AKK bacteria. Searching a new AKK bacterium high-throughput screening method has important significance for rapidly and accurately screening AKK bacterium. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a fluorescent labeling antibody aiming at AKK bacteria Amuc-1100 protein. The second object of the invention is to provide a method for microfluidic high-throughput screening of AKK bacteria. The technical scheme of the invention is summarized as follows: A fluorescent-labeled antibody against AKK bacteria Amuc-1100 protein, prepared by the steps of: Adding PBS buffer solution into an antibody aiming at AKK bacteria Amuc-1100 protein to prepare an antibody solution, adding fluorescein isothiocyanate into dimethyl sulfoxide to prepare fluorescein isothiocyanate-dimethyl sulfoxide solution, adding the fluorescein isothiocyanate-dimethyl sulfoxide solution into the antibody solution, shaking, mixing uniformly, incubating, eluting with reference to an antibody labeling kit instruction book, and purifying to obtain a fluorescence labeling antibody aiming at AKK bacteria Amuc-1100 protein, which is called as fluorescence labeling antibody for short; The antibody against AKK bacterium Amuc-1100 protein comprises a heavy chain variable region and a light chain variable region; HCDR1 in the heavy chain variable region 3 Has the amino acid sequence shown in SEQ ID NO.13 Is shown in the figure; LCDR1 in the light chain variable region 3 With the amino acid sequence shown in SEQ ID NO. 911. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 4. The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 12. A microfluidic high-throughput screening method for AKK bacteria comprises the following steps: The method comprises the steps of 1) preparing a microfluidic chip, wherein the microfluidic chip comprises a square-shaped liquid drop generating channel (3), the middle part of the left channel of the square-shaped liquid drop generating channel (3) is connected with a mobile phase sample injection pool (1), the middle part of the right channel of the square-shaped liquid drop generating channel (3) is connected with a disperse phase sample injection pool (2), the middle part of the right channel of the square-shaped liquid drop generating channel (3) is connected with an S-shaped liquid drop circulating channel (4) through a channel and then is connected with a liquid drop culture pool (5) through a channel, and the liquid drop culture pool (5) is connected with a liquid drop collecting pool (6) through a channel; 2) Collecting fresh feces of healthy volunteers, performing 10 -3-10-5 gradient dilution in an anaerobic incubator by using sterilized normal saline to obtain a diluent, diluting the diluent by using a modified brain heart infusion broth culture medium, and adding the fluorescent-labeled antibody according to one of claims 1-3 to obtain a dispersed phase; 3) Under anaerobic culture conditions, taking mixed liquid of paraffin oil and Span-80 as a mobile phase, introducing the mobile phase into a mobile phase sample injection pool (1) of a microfluid