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Preparation method and antibacterial application of plesiomonas shigelloides phage endolysin

A technology of phage lysing enzyme and Pseudomonas, which is applied in the field of bioengineering, can solve problems such as eye and respiratory tract irritation, complicated production, and pulmonary edema, and achieve the effect of short action time, broad antibacterial spectrum, and wide action spectrum

Active Publication Date: 2016-05-04
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although chlorine dioxide has a good bactericidal effect, it is highly irritating. After contact, it mainly causes eye and respiratory tract irritation. Inhalation of high concentrations can cause pulmonary edema, which can be fatal.
Patent CN101475879B A bactericidal skin care hand sanitizer uses p-chloro-m-xylenol as a bactericide, which also has the disadvantages of strong irritation and toxicity
In order to overcome the shortcomings of chemical fungicides, the patent CN101966134B discloses a bactericidal skin care hand sanitizer and its preparation method, which uses paraben as a fungicide, but the solubility of paraben in water is extremely low, and the product has uniformity. and poor stability
Invention patent CN102600053B Chinese herbal medicine-inorganic antibacterial agent composite sterilizing hand sanitizer and its preparation. Water extracts of mugwort leaves, artemisia annua, baicalin, pomegranate peel, and licorice are used as composite Chinese herbal antibacterial agents, and the inorganic antibacterial agent nano-silver oxide is used, but the production is complicated , low antibacterial power, in order to prevent corruption and deterioration, it needs to be sterilized by autoclaving

Method used

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  • Preparation method and antibacterial application of plesiomonas shigelloides phage endolysin
  • Preparation method and antibacterial application of plesiomonas shigelloides phage endolysin
  • Preparation method and antibacterial application of plesiomonas shigelloides phage endolysin

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Embodiment 1

[0020] Extraction of phage genome

[0021] (1) Crude phage particles

[0022] (1) Preparation of host bacteria: pick Plesiomonas Shigelloides. from solid medium [1] A single colony was inoculated in 5mL LB liquid medium, and cultured with shaking at 37°C for 6-8h.

[0023] (2) Preparation of pure phage culture solution: pick a single phage plaque, inoculate it in 5mL logarithmic phase host bacteria culture solution, culture it with shaking at 37°C for 4-6h, then centrifuge the lysate at 10000rpm for 10min, and the supernatant is ready For the pure culture medium of bacteriophage.

[0024] (3) Preparation of crude phage particles: Transfer the overnight culture of P. shigella-like to 100 mL liquid LB medium, the inoculum size is 1%, and the expansion culture is reached to the logarithmic phase (OD 600 About 0.4), add 5 mL of P. shigella-like phage pure culture solution, shake culture at 37° C. for 6-8 hours to obtain phage lysate. Add DNaseI and RNaseA to the lysate to a fi...

Embodiment 2

[0035] Cloning of lyase gene gp2 and construction of expression vector

[0036] (1) Design a pair of specific primers according to the sequence encoded by the gp2 gene:

[0037] gp2F:5'-CGG GGTACC ATGCAACCATCGCGAGCGTG-3', see SeqID NO.3; gp2R: 5'-CCC AAGCTT CTGGCGGCGGTGGATTTTTG-3', see SeqID NO.4; use the phage genome DNA as a template to amplify the gp2 gene with the above primers, electrophoresis on 1% agarose, and identify the size of the amplified fragment.

[0038](2) The PCR product was directly digested with restriction endonucleases KpnI and HindIII, 37 ° C water bath for 2 hours, 1% agarose electrophoresis, and the gel recovery kit was used to purify and recover the fragments, and the fragments were combined with KpnI and HindIII before The double digested and purified pQE30 vector was ligated overnight at 16°C, and transformed into E. coli M15 / pREP4 competent the next day. The transformation mixture was added to an appropriate amount of LB medium, and incubated ...

Embodiment 3

[0042] Induced expression and purification of lyase gp2 protein

[0043] Inoculate the bacteria HeL1 containing the recombinant plasmid into the LB medium containing ampicillin (100 μg / mL) and kanamycin (25 μg / mL), and cultivate overnight at 37°C with shaking; the next day, transfer to In 100mL LB medium, shake culture at 37°C until OD 600 When the value is about 0.6, add IPTG to a final concentration of 0.5mmol / L, and induce at 16°C for 16h. Bacteria were collected, cells were disrupted by ultrasonic waves, centrifuged at 10,000 rpm / min at 4°C for 10 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter membrane, and the protein expression in the lysed supernatant was analyzed by SDS-PAGE. The filtered lysed supernatant was purified with a His affinity chromatography nickel column (GE Healthcare, Sweden), specifically according to the kit instructions, and the obtained protein was named lyase Gp2.

[0044] The results of SDS-PAGE anal...

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Abstract

The invention relates to a preparation method and antibacterial application of plesiomonas shigelloides phage endolysin. The amino acid sequence of the plesiomonas shigelloides phage endolysin is Seq ID NO.2, and the plesiomonas shigelloides phage endolysin can be prepared into medicine inhibiting bacterial contamination or overgrowth and is applied as bactericide. According to the endolysin, amido bonds between saccharide and peptide of bacterial cell walls or linked bonds between amino acid residues in peptide are hydrolyzed to achieve lysis of host cells finally. The endolysin can act on a host specifically within a short action time and has a wider action spectrum than phage. The endolysin has the advantages of being efficient, specific, free of resistance and the like, and successful application of the endolysin has been reported in the food industry and the pharmaceutical industry at present.

Description

technical field [0001] The design of the invention belongs to the field of bioengineering, and in particular relates to a P. shigella-like phage lyase (Endolysin) and its application as a bactericidal active component in medical hygiene, food processing and hygiene products. Background technique [0002] Phages are viruses that infect prokaryotic microorganisms such as bacteria. Mainly composed of nucleic acids and proteins. The genetic material of the phage is nucleic acid, and the protein constitutes the capsid of the phage. Phage has a simple structure and small size, and can pass through bacterial filters. It is a non-cellular microorganism that strictly parasitizes living cells. Phage synthesizes its own protein and nucleic acid by using the raw materials of the host bacteria, assembles in the host cell, lyses the cell, and releases the progeny phage. [0003] With the improvement of people's living standards, people's demand for hand sanitizers is no longer just for...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N1/21A61K8/66A61Q19/10C12R1/19
CPCA61K8/66A61K2800/85A61Q19/10C12N9/88
Inventor 杨洪江何洋荆兆元
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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