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A coliphage dy1 and its application

A technology of Escherichia coli and bacteriophage, which is applied in the field of bioengineering to achieve the effects of good acid-base and temperature tolerance, fast and efficient lysis, and good stability

Active Publication Date: 2022-05-17
JINAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, pathogenic Escherichia coli can be divided into enteropathogenic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli, Shiga toxin-producing Escherichia coli (including Enterohemorrhagic Escherichia coli) and enteroaggregative Escherichia coli, etc., some serotypes of Escherichia coli of less concern, such as three strains of serotype O34 found in the feces of farmed pigs and cattle in China Escherichia coli, which cannot be lysed well by existing phages

Method used

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  • A coliphage dy1 and its application
  • A coliphage dy1 and its application
  • A coliphage dy1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the separation and purification of phage

[0034] The water samples used to isolate phages in the present invention were collected in Pearl River, Guangzhou in 2018.

[0035] The collected water samples were centrifuged at 8,000g for 10 minutes and then filtered to remove impurities; solid MgSO was added 4 To a final concentration of 50mM. The phage particles were then retained by membrane filtration. Cut the filter membrane into small pieces and put it into 50 mL of eluent (1% beef extract, 3% Tween 80); then take 2.5 mL of eluate and 2.5 mL of TSB medium (1 mM CaCl 2 ) were mixed, and 100 μL of E. coli 180722 bacteria solution was added and shaken overnight at 37°C. After the overnight culture solution was centrifuged at 8,000g for 1 min, the supernatant was filtered, and the filtrate was the stock solution intended to contain phage. Use the double-layer plate method to identify whether there are phages in the filtrate. Take 100 μL of the host bact...

Embodiment 2

[0037] Embodiment 2, the morphological observation of bacteriophage

[0038] Drop the phage suspension on the copper grid, let it settle naturally for 15 minutes, absorb the excess liquid from the side with filter paper, add a drop of 2% phosphotungstic acid on the copper grid to stain the phage for 10 minutes, then absorb the staining solution with filter paper, and wait for the sample to dry Phage morphology was observed with an electron microscope. Transmission electron microscope results showed that ( figure 2 ) The phage has a polyhedron head and a short tail structure, and the diameter of the head is about 54nm. The present invention named it phage DY1. The inventor deposited the phage DY1 in the Culture Collection Center of the Guangdong Provincial Institute of Microbiology, and its preservation number is: GDMCC 61175-B1.

Embodiment 3

[0039] Example 3, phage genome analysis and identification

[0040] Whole-genome sequencing and analysis of phage DY1: the sample DNA was sequenced using the Ion torrent S5 platform, a 500bp library was constructed, and then the optimized sequence was spliced ​​using the SPAdes v.3.6.2 splicing software. And use Prokka1.1.3 to annotate the assembly results, and use NCBI BLASTp to re-annotate the hypothetical protein. Then, the similarity between the phage and the reported phage was determined by NCBI BLASTn, and the genome was compared with the reported similar phage genome according to the annotated protein function, and the comparison results were visualized using Esayfig2.3.3.

[0041] The Genkbank accession number of the genome is: MT808983.1, and the whole genome analysis shows that the genome of phage DY1 is a linear double-stranded structure, the whole genome is 39,817bp long, and 54 open reading frames (Open Reading Frame, ORF) are predicted (Table 1) . ORF25 was p...

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Abstract

The invention provides a coliphage phage, characterized in that, the phage is coliphage DY1, its preservation number is: GDMCC 61175-B1, and the phage DY1 has a polyhedral head and a short tail structure, the head The diameter is about 54 nm, and the Escherichia coli killed by the phage DY1 is serotype O34 Escherichia coli. The phage DY1 in the present invention has good stability at a pH range of 5.0-11 and a temperature of 20-50°C, does not carry any virulence or antibiotic resistance genes, and can be safely used in the prevention and control of Escherichia coli. At the same time, it also has multiple different HNH homing endonucleases, which provide a source for the development of new molecular genetic manipulation tools.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a bacteriophage aimed at Escherichia coli with serotype O34 and an application thereof. Background technique [0002] Escherichia coli is widely distributed in the intestines of humans and different animals, among which, pathogenic Escherichia coli can cause diseases, clinical symptoms such as diarrhea, and severe cases can cause death. At present, antibiotics are the main means of treating E. coli infection. However, according to the 2018 National Bacterial Resistance Surveillance Report, the national average level of E. coli resistance to third-generation cephalosporins is 53%, and the average level of resistance to quinolones is was 50.8%; more than half of E. coli were resistant to these commonly used antibiotics. In addition, polymyxin, known as the last line of defense against multidrug-resistant Gram-negative bacteria in clinical practice, has also been reported ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A01N63/40A01P1/00C12R1/92
CPCC12N7/00A61K35/76A61P31/04A01N63/40C12N2795/10221C12R2001/91C12N1/00Y02A50/30
Inventor 丁郁袁晓鸣吴清平王涓李淳李娜张淑红张菊梅曾海燕
Owner JINAN UNIVERSITY
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