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Enterococcus faecalis phage and separation, purification, enrichment and application thereof

A technology of Enterococcus faecalis and bacteriophage, applied in the field of bioengineering, can solve problems such as alveolar bone defect, tooth loss, periapical abscess, etc.

Active Publication Date: 2019-08-16
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, clinically, some patients still have periapical abscess and progressive bone destruction after repeated standard root canal treatments, resulting in alveolar bone defect and tooth loss, that is, refractory periapical periodontitis

Method used

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  • Enterococcus faecalis phage and separation, purification, enrichment and application thereof
  • Enterococcus faecalis phage and separation, purification, enrichment and application thereof
  • Enterococcus faecalis phage and separation, purification, enrichment and application thereof

Examples

Experimental program
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Effect test

experiment example 1

[0034] Experimental example 1 Isolation and purification of Enterococcus faecalis phage

[0035] 1. Preparation of host bacteria: Enterococcus faecalis E.faecalis YN771 was used as the host bacteria for separating phages. This strain was preserved in the China Center for Type Culture Collection on April 19, 2019. Enterococcus YN771, streaked on BHI solid medium, incubated at 37°C for 24 hours, then picked a single colony and inoculated it in 5mL BHI liquid medium, cultured to early logarithmic growth OD 600 0.3, for the isolation of Enterococcus faecalis phage.

[0036] 2. Isolation of bacteriophages: from isolated cultures to early logarithmic growth OD 600 Enterococcus faecalis YN771 with a value of 0.3 was used as the host bacterium. The sewage without chlorine water treatment was retrieved from the sewage outlet of the Stomatology Department of Yan’an Hospital Affiliated to Kunming Medical University as a sample. The supernatant was suction filtered with a 0.45 μm steril...

experiment example 2

[0041] Experimental Example 2 Phage Morphology Observation

[0042] Take 25 μL of pure phage culture solution and add 50 μL of glutaraldehyde with a concentration of 0.5%. Take the mixed solution and drop it in the middle of a copper grid with a carbon film, let it rest for 30 minutes, absorb the excess liquid with absorbent paper, be careful not to break the copper grid, take 2% phosphotungstic acid was dropped on the copper grid, and the mixed solution was dyed for 2 minutes at room temperature, and then the excess dye solution was absorbed with absorbent paper, and the copper grid was dried at room temperature, and then observed by transmission electron microscope.

[0043] An important basis for phage classification is morphology. Because phages are simple in structure and small in size, their morphological structure can only be observed under an electron microscope after the pure culture particles of phages are negatively stained. The phage PEf771 isolated in the experimen...

experiment example 3

[0044] Experimental Example 3 Study on Biological Characteristics of Phage PEf771

[0045] 1. Infection temperature range and optimum growth temperature of bacteriophage PEf771

[0046] Take valence 10 10 100 μL of pfu / mL phage PEf771 pure culture solution and 300 μL of logarithmic growth phase Enterococcus faecalis YN771 suspension, mix well, let stand at 37°C for 10 minutes, take out and add 4mL BHI semi-solid culture at about 40°C and agar concentration of 0.75% After mixing, pour it evenly and quickly on the BHI solid medium, and place it upside down in constant temperature incubators at 4°C, 10°C, 15°C, 20°C, 25°C, 37°C, 42°C and 55°C respectively. ~3d, the formation of phage plaques was observed, and the experiment was repeated three times.

[0047] The infection temperature range of phage PEf771 is shown in Table 1. It can be seen that phage PEf771 can form transparent plaques with clear edges at 20°C to 42°C, but the activity is strongest at 37°C.

[0048] Table 1 P...

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Abstract

The invention relates to enterococcus faecalis phage and separation, purification, enrichment and application thereof, and belongs to the field of bioengineering. The phage is deposited at the China Center for Type Culture Collection with the accession number of CCTCC NO: M 2019276 on April 19, 2019, and has strong lysis effect on enterococcus faecalis. The phage analyzed morphologically by an electron microscope belongs to typical tailed phage and myoviridae, and is named as enterococcus faecalis phage PEf771; the infection temperature range of the phage PEf771 is 20-42 DEG C; the phage PEf771 has activity being 70% or above when pH is 4-8, and the phage PEf771 has poor thermal stability and cannot tolerate high temperature of 60 DEG C or above for a long time. The phage disclosed by theinvention can be used for specifically lysing the enterococcus faecalis. The application example also proves that the phage can be used for bacterial infection caused by the enterococcus faecalis, andis used as a substitute of antibiotics to safely and effectively maintain oral microecology.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an Enterococcus faecalis phage and its separation, purification, enrichment and application, in particular to an Enterococcus faecalis phage for refractory apical periodontitis and its isolation, purification, enrichment and application. Background technique [0002] Bacteriophages are a class of viruses that host prokaryotic microorganisms. They are widely found in the natural environment, with complex and diverse types. There are about 10 viruses on the earth. 30 ~10 32 The number of phages is ten times or even dozens of times that of bacteria. Almost every bacterium has its corresponding phage. Phage has a simple structure, consisting only of capsid protein and its internal genetic material, lacks independent metabolic capacity, and relies on host cell ribosomes and proteins to synthesize various factors, various amino acids and energy required for its own growth and proliferatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02A61K35/76A61P31/04A61P1/02
CPCC12N7/00A61K35/76A61P31/04A61P1/02C12N2795/10121C12N2795/10151
Inventor 魏云林向盈盈李文玉宋飞季秀玲秦堃豪张东芳
Owner KUNMING UNIV OF SCI & TECH
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