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Broad-spectrum escherichia coli phage, composition and kit capable of simultaneously cracking four bacteria, and application of broad-spectrum escherichia coli phage

A technology of Escherichia coli and phage, applied in the direction of viruses/phages, medical raw materials derived from viruses/phages, applications, etc.

Active Publication Date: 2021-04-20
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The above-mentioned technology has the following defects: the research on coliphage mainly focuses on the lytic properties of enterohemorrhagic phages, and there are few reports on the lysis of other genera of bacteria and applications of coliphages

Method used

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  • Broad-spectrum escherichia coli phage, composition and kit capable of simultaneously cracking four bacteria, and application of broad-spectrum escherichia coli phage
  • Broad-spectrum escherichia coli phage, composition and kit capable of simultaneously cracking four bacteria, and application of broad-spectrum escherichia coli phage
  • Broad-spectrum escherichia coli phage, composition and kit capable of simultaneously cracking four bacteria, and application of broad-spectrum escherichia coli phage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Isolation, preparation and purification of coliphage EC35P1

[0069] In this application, the source samples of Escherichia coliphage EC35P1 (Escherichia coliphage EC35P1) were collected from domestic sewage in Jiangning District, Nanjing City, Jiangsu Province, filtered through double-layer filter paper, centrifuged at low speed and normal temperature, and then filtered the supernatant with a 0.22 μm filter membrane.

[0070] Isolation of phage: Take 10mL of filtered supernatant, add it to 10mL 2 times TSB medium, and add 1mL of phage host strain EC35 logarithmic phase bacteria solution at the same time, place it at 37°C for 8 hours, then take the above culture, and run it at 8000rpm Centrifuge for 10 min under conditions, filter the supernatant with a 0.22 μm filter membrane, and set aside. Take 0.5mL of the logarithmic phase bacterial liquid of the phage host bacteria, add it to 5mL, 48°C semi-solid TSB medium, mix well, pour it on the TSA plate, and prepar...

Embodiment 2

[0072] Example 2 Electron Microscopic Observation of Escherichia coli Phage EC35P1

[0073] Take the culture supernatant of bacteriophage EC35P1 prepared in Example 1 for electron microscope observation: take 20 μL sample and drop it on the copper grid, wait for its natural precipitation for 15 minutes, absorb excess liquid from the side with filter paper, add 1 drop of 2% phosphotungstic acid (PTA) Dye on a copper grid for 10 minutes, absorb the dye solution from the side with filter paper, and observe with an electron microscope after drying. The results are shown in Figure 2. The morphology of coliphage EC35P1 was observed under an electron microscope. It was found to be a tailed bacteriophage without folds. It had a polyhedral symmetrical head and a retractable tail. The transverse diameter is about 50-60nm; the length of the tail is about 95-110nm, and the width of the tail is about 15-25nm. Based on its unique size and shape, coliphage EC35P1 is systematically classifie...

Embodiment 3

[0075] Example 3 Preparation of Escherichia coli Phage EC35P1 Particles and Genome Extraction and Sequencing

[0076] Take 100 mL of phage EC35P1 prepared in Example 1, add DNaseI and RNaseA at a final concentration of 1 μg / mL, incubate at 37°C for 60 minutes, add 5.84 g NaCl (final concentration 1 mol / L), dissolve and place in an ice bath for 1 hour. Centrifuge at 11,000 rpm for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube. Solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w / v), and after it was completely dissolved, it was placed in an ice bath for at least 1 h. Centrifuge at 11,000 rpm for 20 minutes at 4°C, resuspend the pellet with a small amount of SM solution, and obtain the phage particle concentrate, which is stored at 4°C until use.

[0077] Phage nucleic acid was extracted and sequenced using λ phage genomic DNA kit. After nucleotide sequencing, the coliphage EC35P1 (Escherichia coliphage EC35P1) has the nucleot...

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Abstract

The invention belongs to the technical field of escherichia coli phage, and particularly relates to a broad-spectrum escherichia coli phage capable of cracking four bacteria and an application of the broad-spectrum escherichia coli phage to sterilization and bacterium prevention. The invention mainly discloses an escherichia coli phage EC35P1 (Escherichia coli phase EC35P1), and the preservation number of the escherichia coli phage EC35P1 is CCTCC M 2020438. The phage is a virulent phage separated from the nature, tests prove that the phage has no toxic effect on normal microbial flora, and DNA of the phage cannot encode virulence genes and is high in stability. The phage is wide in host range, can be used for cracking escherichia coli, shigella, salmonella and enterobacter cloacae, and large-scale industrial production can be realized. The phage provides an excellent strain resource for developing a novel antibacterial preparation, and has a good application and development prospect.

Description

technical field [0001] The invention belongs to the technical field of coliphage research and development, and in particular relates to a broad-spectrum coliphage capable of simultaneously lysing four kinds of bacteria, a composition and an application thereof. Background technique [0002] Escherichia coli belongs to Gram-negative bacteria and is a resident bacterium in human and animal intestines, which can be divided into two categories: pathogenic and non-pathogenic. The antigen composition of Escherichia coli is complex, which can be divided into bacterial antigen (O), flagellar antigen (H) and surface antigen (K). According to different bacterial antigens, Escherichia coli can be divided into more than 200 types, some of which Escherichia coli of special serotypes are pathogenic to humans and animals, especially to infants and young animals, often causing severe diarrhea and sepsis. Escherichia coli is more resistant to heat than other enteric bacteria, and some bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A23K10/18A23L33/135A23B4/22A61K35/76A61P1/16A61P9/00A61P11/00A61P29/00A61P31/04A61P43/00C12R1/92
CPCY02A50/30
Inventor 樊小九乔欢徐旭凌丛郁何四龙丁良许小康
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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