Method for separating and purifying phage in phage lysate by two-step salting-out extraction

A phage and lysate technology, applied in the field of bioengineering, can solve the problems of phage separation and purification that have not been reported, and achieve good industrial application prospects, shorten operation time, and improve purity

Active Publication Date: 2019-11-22
DALIAN UNIV OF TECH
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  • Application Information

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Problems solved by technology

Compared with the traditional aqueous two-phase system, the salting-out extraction system has the advantages of fast mass transfer, short phase separation time, cheap and easy recovery of solvents, and easy scale-up. It has been widely used in enzymes, proteins, and bio-based chemicals in recent years. , natural products, etc., but there is no report on the isolation and purification of phage

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  • Method for separating and purifying phage in phage lysate by two-step salting-out extraction
  • Method for separating and purifying phage in phage lysate by two-step salting-out extraction
  • Method for separating and purifying phage in phage lysate by two-step salting-out extraction

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preparation example Construction

[0031] Preparation method of phage crude lysate: Klebsiella pneumoniae S2 and phage phiKpS2 were cultured in LB medium (10g / L tryptone, 5g / L yeast extract powder, 10g / L NaCl) and shake flasks for 5-6h Mix according to a certain ratio, and shake flask culture at 37°C and 200rpm. By adjusting the multiplicity of infection (MOI) or co-cultivation time, the crude lysate of phage with different titers can be obtained. The titer of phage, protein concentration , bacteria content and endotoxin content were 10 6 -10 11 pfu / mL, 4-800μg / mL, 10 4 -10 6 cfu / mL, 1.2×10 4 -1.4×10 5 EU / mL.

[0032] 2. Analysis method

[0033] (1) Phage titer determination method:

[0034] Phage titer (Plaque Form Unit) refers to the number of phages in a unit volume sample, which is determined by single-layer agar plate method (spot). Add 1mL of the logarithmic phase host bacterial suspension to the prepared single-layer LB solid medium, spread the bacterial suspension evenly on the solid medium, abs...

Embodiment 1

[0040] The preparation of embodiment 1 phage crude lysate

[0041] Take the Klebsiella pneumoniae KpS2 stored at -20°C, inoculate it in fresh LB medium according to the inoculation amount of 1% (v / v), culture it on a shaker at 37°C and 200rpm for 5-6 hours, and then obtain the logarithmic phase Host bacteria (10 8 -10 9 cfu / mL), and 10 9 The pfu / mL phiKpS2 phage suspension was mixed according to a certain multiplicity of infection (MOI, the number of phages / the number of host bacteria), and then added to fresh LB medium for infection and culture for 2.5-6 hours. In this way, crude lysates of phages with different titers can be obtained, such as a phage titer of 10 6 -10 11 pfu / mL with a total protein content of 400-800 μg / mL and a bacterial concentration of 10 4 -10 6 cfu / mL, endotoxin concentration is 1.2×10 4 -1.4×10 5 EU / mL, pH7.2 or so.

Embodiment 2

[0042] The separation effect of embodiment 2 different organic solvents-dipotassium hydrogen phosphate salting-out extraction system

[0043] The crude lysate of phage that adopts embodiment 1 to make, phage titer is 10 9 pfu / mL, total protein content is 526 μg / mL, OD 650 =0.462, the bacterial concentration is 4×10 5 cfu / mL, the endotoxin concentration is 1.2×10 5 EU / mL, pH7.3. At room temperature of 16°C, add 5.0%, 5.3% and 19.7% of dipotassium hydrogen phosphate to the 50mL centrifuge tube containing the crude lysate of the phage, and gently vortex to mix the salt completely. The total mass of the system is 47.3% of butyl acetate, 44.4% of methyl tert-butyl ether and 35.7% of ethanol, respectively forming three kinds of organic solvents A, B and C - dipotassium hydrogen phosphate salting-out extraction system, centrifuged at 2000g for 10min Accelerate into phase. Take 200 μL of the upper and lower phases, and take out all the agglutinated intermediate phases and dilute ...

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Abstract

The invention belongs to the technical field of biological engineering, and provides a method for separating and purifying phage in a phage lysate by two-step salting-out extraction. The method includes the following steps: dissolving sodium citrate or dipotassium hydrogen phosphate in a phage lysate, adding a lipophilic organic solvent such as ethyl acetate, gently carrying out vortex mixing, andseparating phases to form a three-phase system of top, medium and bottom phases to complete the first step of salting-out extraction; and adding a hydrophilic organic solvent such as n-propanol to the bottom-phase solution obtained in the first step of salting-out extraction, carrying out the second step of salting-out extraction to enrich and purify the phage in the medium phase. The problem that the phage and the bacterial metabolite are hard to separate and the cost is high in the current phage separation process is solved. The method has advantages of simple process, short separation time, high recovery rate, low separation cost and high impurity removal rate, and is a separation method with great industrial application prospects.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for separating and purifying phage in phage lysate by using salting-out extraction technology. Background technique [0002] Since the 21st century, the problem of bacterial resistance caused by the abuse of antibiotics has become a global public health crisis, and the difficulty in the development of new antibiotics has also led to an increasingly severe situation in clinical antibacterial treatment. According to a 2016 British government report, it is estimated that by 2050, the number of deaths caused by "super bacteria" will increase to 10 million people every year. The antibiotic crisis has made the treatment of multi-drug-resistant bacteria and pan-drug-resistant bacteria infection diseases are facing a severe situation where no drugs are available, and it has also made the development of a powerful drug with a large population of indications and significant clinical ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02
CPCC12N7/00C12N2795/00051Y02A50/30
Inventor 修志龙张志荣沈俊涛
Owner DALIAN UNIV OF TECH
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