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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AI-Extracted Technical Summary

Problems solved by technology

There are currently many studies on medical phages, but th...

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.

Application Domain

Recovery/purification

Technology Topic

Multiple drug resistanceBacterial disease +12

Examples

Experimental program(3)

Example Embodiment

[0037] Example 1:

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

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

[0040] (1) Amplify the phage: culture Pseudomonas aeruginosa overnight in a test tube at 35°C and 120 rpm, and expand the culture to the logarithmic phase (OD) in a 250 mL Erlenmeyer flask with an inoculum volume of 1% (v/v) of LB medium 600 About 0.5), add 5g sludge sample (or 5mL sewage), mix 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, inside the flask The medium used is ordinary LB medium;

[0041] (2) Enriching phage: Add ammonium sulfate to a final concentration of 70% (w/v), and let stand at 4°C. The purpose is to precipitate phage particles and enrich the phage. Centrifuge at 10000g for 20min to obtain a precipitate, 70% sulfuric acid Resuspend in ammonium, dialyze in pH 7.4, 0.1mol/L phosphate buffer, use double-layer plate method to detect the number of phages, and set aside;

[0042] (3) Preparation of solution:

[0043] ①0.1mol/L phosphate buffer: add 95mL 0.2mol/L NaH 2 PO4 and 405mL 0.2mol/L Na 2 HPO 4 Mix and dilute to 1000mL.

[0044] ②0.5mol/L sodium hydroxide solution: Weigh 20g of solid NaOH, dissolve it in distilled water, after cooling, dilute to 1000mL.

[0045] (4) Sepharose 4B gel filtration chromatography: Sepharose 4B gel is pre-swelled particles, stored in 20% ethanol, before use, repeatedly washed with distilled water until no ethanol smell; use pH 7.4, 0.1mol/L phosphoric acid Sepharose4B gel was treated with salt buffer, degassed and packed into column (1.0×70.0cm). Equilibrate thoroughly with phosphate buffer; take 1 mL of dialyzed sample and load. Elute at a flow rate of 0.2mL/min, and collect according to the 280nm UV absorption curve and elution volume.

[0046] (5) DEAE-52 anion column chromatography: After DEAE-52 resin is swollen with distilled water, in the order of alkali-acid-alkali, it is treated with 0.5mol/L NaOH solution and 0.5mol/L HCl solution for 30min, and then washed with distilled water to Neutral; DEAE-52 is treated with pH7.4, 0.1mol/L phosphate buffer, degassed and packed into a column (2.6×30cm), fully equilibrated with phosphate buffer; take 5mL gel purified sample on In this way, gradient elution was carried out with a buffer with a limiting concentration of 1mol/L NaCl at a flow rate of 1 mL/min, and collected according to the 280nm ultraviolet absorption curve and elution volume. The calculated phage recovery rate was 70.2%.

[0047] Phage recovery rate (%)=total number of phages in the collection solution of step 5/total number of phages in the enrichment solution of step 2.

Example Embodiment

[0048] Example 2:

[0049] The phage in this example is Staphylococcus aureus phage.

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

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

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

[0053] (3) Preparation of the solution: same as in Example 1;

[0054] (4) Sepharose 4B gel filtration chromatography: same as Example 1;

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

[0056] Phage recovery rate (%)=total number of phages in the collection solution of step 5/total number of phages in the enrichment solution of step 2.

Example Embodiment

[0057] Example 3:

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

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

[0060] (1) Amplify the phage: culture Acinetobacter baumannii in a test tube overnight at 35°C and 120 rpm, expand the culture to the logarithmic phase (OD600 about 0.5) in a 250mL Erlenmeyer flask with 1% inoculum, and add 5g sludge sample (Or 5mL sewage), 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 a 0.22μm filter membrane, the medium used in the triangular flask is ordinary LB medium;

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

[0062] (3) Preparation of the solution: same as in Example 1;

[0063] (4) Sepharose 4B gel filtration chromatography: same as Example 1;

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

[0065] Phage recovery rate (%)=total number of phages in the collection solution of step 5/total number of phages in the enrichment solution of step 2.

PUM

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