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High-temperature-resistant Escherichia coli phage and composition, kit and application thereof

A technology of Escherichia coli and phage, applied in the direction of phage, virus/phage, medical raw materials derived from virus/phage, etc., to achieve good high temperature resistance, high stability, and good application and development prospects

Active Publication Date: 2020-02-28
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, the research on coliphage mainly focuses on the lytic properties of enterohemorrhagic phage, and there are few reports on the stability and application of coliphage

Method used

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  • High-temperature-resistant Escherichia coli phage and composition, kit and application thereof
  • High-temperature-resistant Escherichia coli phage and composition, kit and application thereof
  • High-temperature-resistant Escherichia coli phage and composition, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Isolation, preparation and purification of phage

[0039] The source sample of Escherichia coli phage CL9 in the present invention was collected from the sewage of farmers' market 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.

[0040] Isolation of phage: take 10mL filtered supernatant, add it to 10mL 2 times TSB medium, add 1mL phage host bacteria logarithmic phase bacteria liquid at the same time, place it at 37°C for 6 hours, take the above culture, and put it under the condition of 8000rpm Centrifuge for 10 min, filter the supernatant with a 0.22 μm filter membrane, and set aside. Take 0.5mL of phage host bacteria logarithmic phase bacteria liquid, add 5mL, 48 ℃ semi-solid TSB medium to mix, pour on the TSA plate to prepare a double-layer plate containing the host bacteria. Take 10 μl of th...

Embodiment 2

[0042] Embodiment 2: Electron microscope observation of phage

[0043] Take the supernatant of each phage culture obtained in Example 1 for electron microscope observation: take 20 μ L of 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 ) on a copper grid, dyed for 10 minutes, sucked the dye solution from the side with filter paper, and observed with an electron microscope after drying: the results are as follows: figure 1 As shown, the morphology of Escherichia coli phage CL9 was found to have a polyhedral head and a long tail, the long diameter L is 50-60nm, the transverse diameter W is 50-60nm, L / WY≈1; the tail length is 60-70nm, 2 to 8 nm wide, with contractile muscle sheaths (see figure 1 ).

Embodiment 3

[0044] Example 3: Extraction and sequencing of phage genome

[0045] Take 100 mL of each phage prepared in Example 1, add DNaseI and RNaseA at a final concentration of 1 μg / mL, incubate at 37°C for 60 min, add 5.84 g NaCl (final concentration 1 mol / L), dissolve and place in an ice bath for 1 h. Centrifuge at 11,000 rpm for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube. Add solid PEG8000 (final concentration 10%, that is, add 10 g to 100 mL), and after complete dissolution, ice bath for at least 1 h. Centrifuge at 11,000 rpm for 20 min at 4°C, and resuspend the pellet with a small amount of SM solution. Add an equal volume of chloroform and isoamyl alcohol for extraction, shake gently for 30 s, centrifuge at 8000 rpm for 1 min, absorb the supernatant, and repeat the extraction until clarification. Add DNase I and RNase A again to a final concentration of 1 μg / mL, and react at 37°C for 30-60 minutes. Add EDTA to a final concentration of 20 mmol / L (that ...

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Abstract

The invention belongs to the technical field of Escherichia coli, and particularly relates to a high-temperature-resistant Escherichia coli phage and a composition, a kit and application thereof. Thephage is Escherichia coli phage CL9 which is collected under the number of CCTCC M 2018937. The phage is nonhazardous to normal microbial flora, resistant to high temperature and high in stability andhas good application and development prospect, DNA of the phage cannot code virulence genes, and an excellent strain resource is provided for developing novel antibacterial preparations.

Description

technical field [0001] The invention belongs to the technical field of Escherichia coli research and development, and in particular relates to high temperature-resistant coli phages, compositions, kits and applications thereof. Background technique [0002] Escherichia coli is a Gram-negative bacteria that is resident in the intestinal tract of humans and animals. Most strains of E. coli are harmless. However, some E. coli bacteria can cause foodborne illness. 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. This bacterium is more resistant to heat than other intestinal bacteria, and some bacteria stil...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCC12N7/00A61K35/76A61P31/04C12N2795/10121C12N2795/10132A61K2300/00Y02A50/30
Inventor 赵伟丁良樊小九肖逍许小康丛郁徐旭凌
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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