Method for Separating and Purifying Antimicrobial Peptides from Plant Endophytic Bacteria Strains by High Performance Liquid Chromatography

A technology of high-performance liquid chromatography and plant endophytic bacteria, which is applied in the fields of peptide preparation methods, botany equipment and methods, and microbial-based methods, and can solve problems such as high cost, suicide of host microorganisms, and inability to obtain expression products

Inactive Publication Date: 2016-05-04
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemically synthesized peptides, the cost is high
However, through genetic engineering, direct expression of antimicrobial peptide genes in microorganisms may cause host microorganisms to commit suicide and fail to obtain expression products.

Method used

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  • Method for Separating and Purifying Antimicrobial Peptides from Plant Endophytic Bacteria Strains by High Performance Liquid Chromatography
  • Method for Separating and Purifying Antimicrobial Peptides from Plant Endophytic Bacteria Strains by High Performance Liquid Chromatography
  • Method for Separating and Purifying Antimicrobial Peptides from Plant Endophytic Bacteria Strains by High Performance Liquid Chromatography

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of embodiment 1 bacterial strain B-011 of the present invention

[0029] Rinse the healthy tobacco plant samples collected from the tobacco bacterial wilt disease area in Changsha City, Hunan Province with sterile water, then dry the water with sterilized filter paper, then wash with 70% alcohol, and put the samples on the alcohol lamp to sterilize the surface. Bacteria, cut off the epidermis of the plant stem with a sterile knife, cut a small amount of xylem and put it into a test tube filled with 10ml sterile water. -1 -10 -4 , take 1ml bacterial solution for each gradient and apply it on the BPA plate medium, apply 1 plate for each concentration, repeat 3 times, and culture in a 28-30°C incubator. After culturing for 3 days, pick colonies of different shapes and re-streak on the plate for purification to obtain the strain of the present invention. Finally, transfer it to the inclined plane of the test tube and save it for later use.

Embodiment 2

[0030] Embodiment 2 Preparation of bacterial strain B-011 bacterium fermentation filtrate of the present invention

[0031] Preparation of seed solution: Inoculate the strain of the present invention preserved on a slant into BPY culture solution with a volume of 100mL / 250mL, initial pH 7.0, 30°C, 180r / min shaking culture for 24h.

[0032] Fermentation culture: Inoculate the seed liquid into BPY medium with 1% inoculum amount, the liquid volume is 100mL / 250mL, the initial pH is 7.0, 30°C, 180r / min shaking fermentation culture for 48h.

[0033] Fermentation broth treatment: centrifuge the liquid culture obtained through the above fermentation at room temperature at 10,000 r / min for 10 minutes, and filter the supernatant through a microporous membrane with a pore size of 0.22 μm to obtain a fermentation filtrate containing antibacterial active substances.

[0034] BPY culture medium / medium: peptone 10g, beef extract 5g, yeast extract 5g, NaCl 5g, glucose 5g, water 1000mL, pH7.0....

Embodiment 3

[0035] Embodiment 3 The influence of fermented filtrate of the present invention on tomato early blight spore germination

[0036] Cultivate tomato early blight bacteria on PDA for a certain period of time to produce spores, wash off the spores with a small amount of sterile water, add sterile water to dilute to 40 times objective lens, 40 spores in each field of view. Take the above-prepared spore suspension and add equal volumes of the above-mentioned fermentation filtrates of different concentrations, mix well, take the addition of sterile water as a control, take 10 μL and drop it on the concave glass slide. Incubate with moisture at 28°C for 24 hours, observe the germination of spores, and repeat each treatment 3 times. A spore germ tube whose length is greater than the diameter of the spore is regarded as germinated.

[0037] The results showed that the spores of the control group formed elongated, smooth and uniform germ tubes (see figure 1 C). 50% fermentation filtr...

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Abstract

The invention relates to a method for separating and purifying the antibacterial peptide of an endophytic bacteria strain though high efficiency liquid chromatography, and the method comprises the following steps of: preparing the fermentation filter liquor of the strain by taking brevibacillus brevis B-011 as a basic strain, wherein the strain is collected in the China general microbiological culture collection center in April 12, 2013, and the collection number is CGMCC No.7466; adding acetonitrile; uniformly mixing, and standing; centrifugalizing; evaporating and concentrating a supernatant to obtain an antibacterial crude extract; and then carrying out secondary separation by adopting the high efficiency liquid chromatography to obtain an antibacterial peptide activity single-peak component. According to the invention, a bacteriostatic activity substance ( antibacterial peptide) contained in the fermentation filter liquor of the strain B-011 has inhibiting effect on the spore germination of alternaria solani and shows the teratogenic effect on germ tubes and hyphae. The method disclosed by the invention can be used for separating and purifying the bacteriostatic activity component contained in the strain fermentation filter liquor through the high efficiency liquid chromatography and identifying the substance and is conductive to defining the root cause of the strain on inhibiting the alternaria solani and developing the novel antibacterial drug.

Description

technical field [0001] The invention relates to a method for separating and purifying antibacterial active substance antibacterial peptide, in particular to a method for separating and purifying antibacterial active substance (antibacterial peptide) of plant endophytic bacterial strains by high performance liquid chromatography. Background technique [0002] At present, all conventional antibiotics have corresponding drug-resistant pathogenic strains, and the problem of drug resistance of pathogenic bacteria has become an increasingly serious threat to people's health. Finding new types of antibiotics is an effective way to solve the problem of drug resistance. Antimicrobial peptides are considered to have broad application prospects in the pharmaceutical industry because of their high antibacterial activity, wide antibacterial spectrum, many types, wide range of options, and the lack of resistance mutations in target strains. [0003] However, the main means of obtaining a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/00C07K1/16A01N63/02A01P3/00C12R1/01
Inventor 易有金王宇婷曾静
Owner HUNAN AGRICULTURAL UNIV
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