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A kind of coliphage and its application

A technology of Escherichia coli and bacteriophage, which is applied in the field of bioengineering to achieve strong killing activity, low toxic and side effects, and high safety

Active Publication Date: 2019-11-12
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of phages to disinfect enterohaemorrhagic Escherichia coli (especially mcr-1 positive enterohaemorrhagic Escherichia coli) in the environment

Method used

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  • A kind of coliphage and its application
  • A kind of coliphage and its application
  • A kind of coliphage and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Phage Isolation and Preparation

[0030] Streak-inoculate the host bacteria on 1.2% LB agar medium, culture overnight, pick a single clone and inoculate it in 1ml LB liquid medium, culture it with shaking at 37°C for 5-6 hours, and use it as the host bacteria culture for later use.

[0031] In the summer of 2016, sewage from a goose farm in Changzhou City, Jiangsu Province was collected, and the supernatant was filtered through double-layer filter paper, centrifuged at 10,000rpm for 20min, and then filtered with a 0.22μm filter membrane; 10mL of the filtered supernatant was added to 0.5mL of the host bacteria Overnight culture, followed by sterile CaCl 2 After mixing the mother liquor to a final concentration of 1.25mM, add 20ml of LB liquid medium, react at room temperature for 30min, and place it at 37°C for 6-8h; then take the culture and centrifuge at 12000rpm, 4°C for 30min, and take the supernatant; 10mL of this supernatant was added to 0.5mL of the ove...

Embodiment 2

[0034] Example 2 Phage Amplification and Purification

[0035] On the double-layer plate forming plaques in Example 1, use the tip of a pipette to pick up a single plaque with a larger diameter, inoculate it in 3-5 ml LB liquid medium, and add 0.1 ml of phage host bacterial solution. mL, mix well, act at room temperature for 15 minutes, incubate at 37°C for 10-14 hours, centrifuge at 12,000 rpm, 4°C for 10 minutes, take the supernatant, add 0.3% chloroform; repeat the double-layer experiment, and pick a single phage plaque repeatedly 4-5 times. Purify the phage into plaques of equal size.

[0036] Take 1 mL of freshly cultured host bacteria and add 0.3 mL of phage lysate (the ratio of a single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Incubate at 37°C for 20 minutes to make the phage particles adsorb to the host bacteria; add 100mL LB liquid medium, then add CaCl 2 The mother solution was grown to a final concentration of 1.25mM, shaken at 37°C fo...

Embodiment 3

[0041] Example 3 Transmission electron microscope observation of bacteriophage vB_EcoM_C1

[0042] Take the PEG-purified phage in Example 2 for electron microscope observation. The specific operation steps are: add 10 μL of the sample and drop it on the copper grid, wait for it to settle for 15 minutes, absorb the excess liquid with filter paper, and use 2% phosphotungstic acid (PTA) Stain for 1-2min, observe with a transmission electron microscope (Hitachi H-7650) after drying; the observation results are as follows figure 2 As shown, the head is in the shape of an icosahedron, the diameter of the head is about 80 nm, and the length of the tail is about 110 nm. According to the "Classification of Viruses—The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, vB_EcoM_C1 belongs to the family Myoviridae ( Myoviridae ).

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Abstract

The invention discloses a coliphage B_EcoM_C1 with the preservation number of CCTCC NO:M 2017190. The coliphage B_EcoM_C1 is a double-stranded DNA, belongs to a myovirus family, having a regular dodecahedron-shaped head and a telescopic tail, and can form a transparent and bright plaque on a solid medium, no aureoles exist surrounding the plaque, the plaque has clear and regular edge, and the diameter is 0.5-1 mm. The coliphage B_EcoM_C1 has a good eliminating and killing effect on enterohemorrhagic Escherichia coli in the space environment, and especially mcr-1 positive enterohemorrhagic Escherichia coli, and can be applied to the preparation of medicines for controlling the enterohemorrhagic Escherichia coli infection in order to realize a broad-spectrum sterilization effect.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an enterohemorrhagic coli phage vB_EcoM_C1 and its application. Background technique [0002] Enterohemorrhagic Escherichia coli (EHEC) is a subgroup of Shiga-toxin producing Escherichia coli (STEC), which was confirmed to be an important zoonotic pathogen in 1982 and can It can cause different degrees of diarrhea, hemorrhagic colitis (Hemorrhagiccolitis, HC), hemolytic uremic syndrome (Haemolytic uraemic syndrome, HUS), etc., and severe cases can lead to death. [0003] Enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli) is widely distributed in soil, water, agricultural and forest products in nature, and is also common in the intestinal tract and respiratory tract of humans and animals. It is a typical opportunistic pathogen and is often used in animal resistance Explosive epidemics are caused when the power is reduced, such as goose diarrhea, enteritis of rab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04
CPCA61K35/76C12N7/00C12N2795/10121C12N2795/10131C12N2795/10132Y02A50/30
Inventor 孙利厂王冉葛展霞何涛庞茂达
Owner JIANGSU ACAD OF AGRI SCI
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