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Escherichia coli bacteriophage vBE_coM_swi3 and application thereof

A technology of Escherichia coli and phage, which is applied in the field of bioengineering, can solve the problems of undeveloped phage resources and achieve good application prospects, good physical and chemical factor tolerance, and good safety effects

Active Publication Date: 2021-01-29
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Theoretically, any pathogenic bacteria has its corresponding phage, but the type and quantity of phage we have found so far is only the tip of the iceberg, and there are still a large number of phage resources that have not yet been developed

Method used

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  • Escherichia coli bacteriophage vBE_coM_swi3 and application thereof
  • Escherichia coli bacteriophage vBE_coM_swi3 and application thereof
  • Escherichia coli bacteriophage vBE_coM_swi3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Isolation and preparation of Escherichia coli phage vB_EcoM_swi3

[0045] (1) Experimental materials:

[0046] Fecal sewage samples were collected from pig slaughterhouse sewage in Linyi City, Shandong Province. The phage host bacteria was E. coli-K88, which was isolated, identified and preserved from diseased pig feces by our laboratory.

[0047] (2) Experimental method:

[0048] Add 5ml of the collected sewage sample to 50ml of LB liquid medium, then add 500μL of Escherichia coli E.coli-K88 bacterial solution, mix well and put it into a 37°C constant temperature incubator for overnight cultivation. The culture was first coarsely filtered with four layers of gauze, the filtrate was centrifuged at 4000rpm for 10min, the supernatant was centrifuged at 12000rpm for 15min, and then the supernatant was filtered with a 0.22μm filter to obtain the phage stock solution. Spread 200 μL of Escherichia coli E.coli-K88 proliferation solution evenly on the surface of LB...

Embodiment 2

[0049] Example 2: Purification and Mass Propagation of Escherichia coli Phage vB_EcoM_swi3

[0050] Q1. Purification of coliphage vB_EcoM_swi3

[0051] Purify the samples identified to contain phages in the above Example 1. The specific operation is as follows: Take 100 μL of the phage stock solution and perform a 10-fold dilution. Take 100 μL of the phage stock solution at each dilution and mix it with an equal amount of overnight cultured E.coli-K88. Add 3ml of 0.7% LB semi-solid medium heated to 50°C and mix well, pour it into the upper layer of the LB agar plate, let it stand for 5 minutes, wait for it to solidify, and culture it upside down at 37°C for 4-5 hours to obtain the formation of phage plaques double-layer flat panel. Pick out a single phage plaque, add 3ml of LB liquid medium, bathe in 40°C water for 30min, centrifuge at 12000rpm for 5min, take the supernatant and filter it with a 0.22μm filter to obtain the phage filtrate. Take 100 μL of phage filtrate and di...

Embodiment 3

[0056] Example 3: Morphological Observation by Transmission Electron Microscopy of Escherichia coli Phage vB_EcoM_swi3

[0057] Take 20 μL of the purified phage proliferation solution and drop it on the copper grid, let it stand for about 15 minutes, and absorb the excess liquid with filter paper. Add 15 μL of 2% phosphotungstic acid (PTA) dropwise on the copper mesh for 5 minutes, absorb excess dye solution with filter paper, and observe with a transmission electron microscope after drying.

[0058] The transmission electron microscope results of coliphage vB_EcoM_swi3 are as follows figure 1 As shown, the coli phage vB_EcoM_swi3 has an obvious regular polyhedral head structure and a stretchable tail structure. The diameter of the head is about 80 nm, and the length of the tail is about 120 nm. According to the "Classification of Viruses-The Tenth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (IC...

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Abstract

The invention discloses an escherichia coli bacteriophage vBE_coM_swi3 with a preservation number of CCTCC NO: M2019467. The bacteriophage belongs to myoviridae and has a polyhedral head and a telescopic tail structure, the diameter of the head is about 80 nm, and the length of the tail is about 120 nm; transparent empty spots can be formed on a solid culture medium, no halo ring exists around thesolid culture medium, the edge is clear and regular, and the diameter is about 1-1.5 mm; the bacteriophage has a good cracking effect on swine-derived and chicken-derived escherichia coli in a breeding environment, particularly on swine-derived and chicken-derived pathogenic escherichia coli, and has very wide application prospects in multiple aspects of pharmaceutical preparations, feed additives and the like for preventing and treating swine colibacillosis and chicken colibacillosis.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a coli phage vB_EcoM_swi3 and its application. Background technique [0002] Escherichia coli is an opportunistic pathogen, and the pathogenicity is related to the serotype of Escherichia coli, and there are differences among different animals. Among them, porcine colibacillosis and chicken colibacillosis are widespread in the breeding environment. Once infected, they will cause diarrhea, significantly affect the weight gain of animals, and cause the mortality rate of animal groups to rise sharply. Diseased animals can also pass through feces, etc. coli from pigs and chickens in the breeding environment will cause continuous infection of animal populations, causing great economic losses to farmers. [0003] At present, the use of antibiotics is the main means of preventing and treating colibacillosis in farms. However, due to the extensive abuse of antibiotics, it has d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A23K10/18A23K50/30A23K50/75A01N63/40A01P1/00C12R1/92
CPCA23K10/18A23K50/30A23K50/75A01N63/40C12N2795/00021C12N2795/00031
Inventor 张灿韩丽丽任慧英刘文华
Owner QINGDAO AGRI UNIV
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