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A Broad Spectrum Escherichia coli Phage and Its Application

A technology of Escherichia coli and bacteriophage, applied in the direction of bacteriophage, virus/phage, application, etc., can solve the problems of medical and health industry crisis, increased expenditure and other problems, and achieve the effect of wide host range, excellent strain resources, and good application development prospects.

Active Publication Date: 2022-05-17
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment of MDR (multidrug resistant) bacteria and ESKAPE (Enterococcusfaecium, Klebsiella penumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Enlerobacterspp.) pathogenic bacteria not only increases related expenditures, but also brings potential crises to the medical and health industry

Method used

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  • A Broad Spectrum Escherichia coli Phage and Its Application
  • A Broad Spectrum Escherichia coli Phage and Its Application
  • A Broad Spectrum Escherichia coli Phage and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Isolation and Purification of Escherichia coli Phage WXLECP008

[0045] Collect 10 samples of sewage near a chicken farm in Hubei, each 20mL, centrifuge at 5000rpm for 10min, take 10mL supernatant and sterilize it, mix it with 10mL 2×TSB liquid medium and 2mL Escherichia coli WXLEC008 in logarithmic phase Liquid (10 8 cfu / mL) were evenly mixed, and cultured overnight at 180 rpm at 37°C to enrich the phage. The sample enrichment solution was centrifuged at 5000 rpm for 10 min, and the supernatant was sterilized through a 0.22 μm microporous membrane to obtain a filtrate containing phage. Take 100 μL of the filtrate and mix it evenly with 300 μL of the host Escherichia coli liquid, and let it stand for 15 minutes to fully bind with the receptors on the surface of the bacteria. Add the above mixed solution to 4mL TSB semi-solid agar medium cooled to 50°C, spread it on the solidified TSA plate immediately after mixing, and incubate upside down at 37°C for 8-12 h...

Embodiment 2

[0046] Example 2 Determination of coliphage WXLECP008 titer

[0047] Make diluent with SM liquid, the stoste 10 times gradient of coliphage WXLECP008 (made by embodiment 1) is diluted step by step to 10 8 times. Take l0 respectively 5 , l0 6 , l0 7 and l0 8 0.1 mL of the diluted phage culture solution was evenly mixed with 500 μL of the host bacterium (Avian Nonpathogenic Escherichia coli (ANPEC)) Escherichia coli WXLEC008, and allowed to stand for 15 minutes to allow it to fully bind to the receptors on the surface of the bacteria. Add the above mixed solution to 4 mL of TSB semi-solid agar medium cooled to 50°C, mix well and spread it on the solidified solid agar plate immediately, and incubate it upside down at 37°C for 8-12 hours after the agar solidifies. Three parallel samples are required for each dilution, and the average of the three parallel samples of this dilution is used for counting. Among them, phage titer (PFU / mL) = average number of plaques × dilution fa...

Embodiment 3

[0051] Example 3 Determination of coliphage WXLECP008 to the optimal multiplicity of infection (MOI) of Escherichia coli

[0052] A single colony of Escherichia coli was picked, inoculated into a bacterial bottle containing 3 mL of TSB culture solution, and shaken at 180 rpm in a shaker at 37°C for 12 hours to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL TSB culture medium at a ratio of 1:100, and cultured with shaking at 180 rpm at 37°C to the pre-logarithmic phase. Add the pure culture solution of phage WXLECP008 (prepared in Example 1) and host bacteria (MOI = number of phages / number of bacteria) according to the multiplicity of infection ratio, and add TSB liquid medium to make the total volume of each tube the same. Shake overnight at 180 rpm in a shaker at 37°C. After the culture was completed, centrifuge at 5000 g for 10 min and collect the supernatant to measure the phage titer. Each point was cultured in duplicate and the ave...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a broad-spectrum colibacillus phage and its application. The Escherichia coli phage WXLECP008 (Escherichia coli phage, WXLECP008) provided by the present invention is a virulent phage, which has tolerance at pH 4 to pH 10 and at -80°C to 65°C, cultivated for 8 hours under the condition of MOI=0.1, and its titer is 3.5×10 10 pfu / mL. The Escherichia coli phage provided by the invention is a broad-spectrum phage with a wide cleavage range, has high titer and high safety, can reproduce in large quantities in non-pathogenic host bacteria, and can be used for large-scale fermentation production. The phage described in the invention can provide excellent bacterial strain resources for the development of phage therapy, has good application and development prospects, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a broad-spectrum colibacillus phage and its application. Background technique [0002] Escherichia coli (Escherichia coli), commonly known as Escherichia coli, has been regarded as a component of normal intestinal flora for quite a long time and is considered to be a non-pathogenic bacterium. It was not until the middle of the 20th century that it was recognized that some special serotypes of Escherichia coli were pathogenic to humans and animals, often causing severe diarrhea and sepsis. Therefore, most E. coli are generally regarded as opportunistic pathogens. According to different biological characteristics, pathogenic Escherichia coli can be divided into 6 categories: enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterohaemorrhagic Escherichia coli (EHEC), enteroadhesive Escherichia coli (...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04A01N63/40A01P1/00C02F3/34C12R1/92
CPCC12N7/00A61K35/76A61P31/04A01N63/40C02F3/34C12N2795/10121C12N2795/10132C12N2795/10131Y02A50/30
Inventor 王喜亮吴泳茵李越赵虹泽吕佩琳唐路遥金秀娥
Owner HUAZHONG AGRI UNIV
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