Method for purifying phage through chromatography

A purification method and phage technology, applied in the biological field, can solve the problems of no related research reports on phage purification, and achieve the effect of overcoming the poor curative effect of drugs and treating bacterial diseases

Inactive Publication Date: 2010-12-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently many studies on medical phages, but there are no relevant research reports on phage purification

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The phage in this example is Pseudomonas aeruginosa phage.

[0039] A phage chromatography purification method, the specific method is:

[0040] (1) Amplify phage: 35 DEG C, under the condition of 120rpm, cultivate Pseudomonas aeruginosa overnight in the test tube, expand the culture to the logarithmic phase (OD 600 About 0.5), add 5g of sludge sample (or 5mL of sewage), after mixed culture for 6h, centrifuge at 8000g for 10min, remove most of the bacteria and sample impurities, collect the supernatant, filter with a 0.22μm filter membrane, and put it in the Erlenmeyer flask The medium used is common LB medium;

[0041] (2) Enrichment of phage: add ammonium sulfate to a final concentration of 70% (w / v), let it stand at 4°C, the purpose is to precipitate the phage particles, enrich the phage, and centrifuge at 10000g for 20min to obtain the precipitate, 70% sulfuric acid Resuspended in ammonium, dialyzed in pH 7.4, 0.1mol / L phosphate buffer, detected the number of phag...

Embodiment 2

[0049] In this example, the phage is Staphylococcus aureus phage.

[0050] A phage chromatography purification method, the specific method is:

[0051] (1) Amplify phage: 35 DEG C, under the condition of 120rpm, cultivate Staphylococcus aureus overnight in the test tube, expand the culture to the logarithmic phase (OD600 approx. 0.5), add 5g of sludge sample (or 5mL of sewage), after mixed culture for 6h, centrifuge at 8000g for 10min to remove most of the bacteria and sample impurities, collect the supernatant, filter and sterilize with 0.22μm filter membrane, the culture used in the Erlenmeyer flask Base is common LB culture medium;

[0052] (2) Enriched phage: same as Example 1;

[0053] (3) preparation of solution: with embodiment 1;

[0054] (4) Sepharose 4B gel filtration chromatography: with embodiment 1;

[0055] (5) DEAE-52 anion column chromatography: the method is the same as in Example 1, and the calculated phage recovery rate is 68.5%.

[0056] Phage recovery...

Embodiment 3

[0058] The phage in this example is Acinetobacter baumannii phage.

[0059] A phage chromatography purification method, the specific method is:

[0060] (1) Amplified phage: 35°C, 120rpm, culture Acinetobacter baumannii overnight in a test tube, expand the culture to logarithmic phase (OD600 about 0.5) in a 250mL Erlenmeyer flask with 1% inoculum, add 5g sludge sample (or 5mL sewage), after 6 hours of mixed culture, centrifuge at 8000g for 10 minutes to remove most of the bacteria and sample impurities, collect the supernatant, filter and sterilize with a 0.22μm filter membrane, and the medium used in the triangular flask is ordinary LB medium;

[0061] (2) Enriched phage: same as Example 1;

[0062] (3) preparation of solution: with embodiment 1;

[0063] (4) Sepharose 4B gel filtration chromatography: with embodiment 1;

[0064] (5) DEAE-52 anion column chromatography: the method is the same as in Example 1, and the calculated phage recovery rate is 78.3%.

[0065] Phage...

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PUM

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Abstract

The invention relates to a method for purifying phage through chromatography, which comprises the following steps of: (1) amplifying the phage; (2) enriching the phage to obtain a phage suspension; and (3) performing column chromatography: performing column chromatography to the enriched phage to obtain the purified phage, wherein a column packing material for column chromatography is sepharose 4B gel or DEAE-52 resin. The purified phage can be applied to clinic treatment and can overcome the defect of poor drug curative effect caused by antibiotic abuse and production of pathogenic drug-resisting stains. Under the current situation that multiple drug-resisting stains widely exist and no effective treatment method is available, the phage can be used for cracking pathogenic bacteria and killing host cells, and thus, the purpose of treating bacterial disease is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the purification of phages, in particular to a purification method of phages by chromatography. Background technique [0002] As a fast and effective drug, antibiotics have been widely used in the treatment of bacterial diseases for a long time, resulting in the continuous emergence of drug-resistant pathogenic bacteria, and the abuse of antibiotics has led to the continuous emergence of multi-drug-resistant pathogenic bacteria, which has brought new challenges to clinical treatment. come with great difficulty. Bacteriophage (phage) is a general term for viruses that can infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes. Some of them can cause lysis of host bacteria, so they are called phages. Because of their ability to lyse drug-resistant pathogenic bacteria, phages are among the most likely candidates for antimicrobials. After the phage specifically bind...

Claims

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Application Information

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IPC IPC(8): C12N7/02
Inventor 杨洪江梁莉李玲艳张晓梅朱会霞
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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