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Pseudomonas aeruginosa phage lyase and application

A technology of Pseudomonas aeruginosa and phage lysing enzymes, applied in the field of bioengineering, can solve problems such as hazards and potential hazards, change water taste, equipment corrosion, etc., achieve good water solubility, improve bactericidal activity, and prevent pollution.

Active Publication Date: 2017-08-18
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical cleaners can efficiently remove biofilms, but they bring certain hazards and potential hazards to the human body, bring certain corrosiveness to equipment, and at the same time, cause certain environmental protection problems to the environment
For example, the chlorine contained in cleaning agents will change the taste of water and cause certain negative effects on the human body and the environment; bacteriophages are considered to be a new and effective method to control biofilms, but there are many disadvantages in the process of using phages, for example, against Different biofilms require different phages, and the host range of phages is narrow, and bacteria that are not sensitive to phages cannot be eliminated; the cracking ability is not strong; they have a screening effect on bacteria; and there are certain risks in the use of phages

Method used

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  • Pseudomonas aeruginosa phage lyase and application
  • Pseudomonas aeruginosa phage lyase and application
  • Pseudomonas aeruginosa phage lyase and application

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preparation example Construction

[0045]The present invention provides a preparation method for Pseudomonas aeruginosa phage lyase, using primer 5'-GAGCTCATGGGATCCTTCTTCGTAGCACCGGGCTCCTCCGCTCTAACTGAGCAAG-3' and primer 5'-CTGCAGTTACTTGAAGGATTGATAGG-3'; -3'; Amplify the artificial Pseudomonas aeruginosa phage lyase gene from the Pseudomonas aeruginosa phage genomic DNA; the specific steps are as follows

[0046] (1) Amplifying the artificial Pseudomonas aeruginosa phage lyase gene from the Pseudomonas aeruginosa phage genome;

[0047] (2) Construction of a recombinant expression vector expressing artificial Pseudomonas aeruginosa phage lyase;

[0048] (3) Transform the recombinant expression vector into Escherichia coli competent cells, and screen to obtain engineering bacteria expressing artificial Pseudomonas aeruginosa phage lyase;

[0049] (4) Isopropyl-β-D-thiogalactopyranoside induces expression to obtain recombinant gene expression products;

[0050] (5) The recombinant gene expression product is purifi...

Embodiment 1

[0052] The extraction of embodiment 1 phage genome

[0053] (1) Crude phage particles

[0054] (1) Preparation of host bacteria: Pick a single colony of Pseudomonas aeruginosa from the solid medium and inoculate it in 5mL LB liquid medium. Shake culture for 6-8h.

[0055] (2) Preparation of phage pure culture medium: pick a single phage plaque and inoculate it in 5mL logarithmic phase host bacteria culture medium, Shake culture for 4-6 hours, then centrifuge the lysate at 10000×g for 10 minutes, and the supernatant is the pure phage culture solution.

[0056] (3) Preparation of crude phage particles: Transfer the overnight culture to 100mL liquid LB medium with an inoculum size of 1%, amplify and culture to the logarithmic phase (OD 600 About 0.4), add 5mL phage pure culture solution, The phage lysate was obtained after shaking culture for 6-8 hours. Add DNase I and RNase A to the lysate to a final concentration of 5 μg / mL, mix well Let stand for 1h. Then add NaCl t...

Embodiment 2

[0066] The construction of embodiment 2 recombinant plasmids

[0067] 1. Obtaining the target fragment

[0068] ⑴ Primer design

[0069] According to the lyase gene sequence (Seq ID NO.2), design primers:

[0070] HPP-Lysgp57-F: 5'-GAGCTCATGGGATCCTTCTTCGTAGCACCGGGCTCCTCCGCTCTAACTGAGCAAG-3'

[0071] HPP-Lysgp57-R: 5'-CTGCAGTTACTTGAAGGATTGATAGG-3'

[0072] PCNP-Lysgp57-F: 5'-GCGGGATCCATGAAACGCAAGAAACGTAAGAACGCAAAGCTCTAACTGAGCAAG-3'

[0073] PCNP-Lysgp57-R: 5'-CGGGGTACCTTACTTGAAGGATTGATAGG-3';

[0074] (2) 50μL reaction system:

[0075]

[0076]

[0077] (3) PCR reaction conditions:

[0078]

[0079] 2. Chemical transformation experiment (construction of T-easy recombinant vector)

[0080] (1) Ligate the gp57 gene PCR product with the T-easy vector, the connection system is:

[0081] 10μL reaction system:

[0082]

[0083] Mix the reaction system, Connect overnight.

[0084] ⑵Escherichia coli DH5a chemically competent cell preparation:

[0085] a. Bacteri...

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Abstract

The invention relates to pseudomonas aeruginosa phage lyase and application. The artificial pseudomonas aeruginosa phage lyase has nucleic acid sequences 1 and 2 and amino acid sequences 3 and 4; a gene engineering technology is utilized and polycation nonapeptide and hydrophobic pentapeptide are added at an N end of previous Gram-negative bacterium phage lyase and are developed to obtain artificial lyase capable of efficiently killing pseudomonas aeruginosa, pseudomonas putida, bacillus licheniformis and bacillus subtilis. The lyase and a derivative thereof can be independently used or can be coordinated with a surfactant and can be used for specifically deactivating the pseudomonas aeruginosa and bacillus; the lyase can be used for removing and inhibiting a biological coating film formed by microorganisms, and an enzyme preparation source which is safe and has no toxic or side effect is provided for preventing and controlling infection of the pseudomonas aeruginosa, the bacillus licheniformis and the bacillus subtilis at present and controlling biological coating film pollution caused by bacteria including the bacillus licheniformis and the bacillus subtilis and the like in foods.

Description

technical field [0001] The design of the invention belongs to the field of bioengineering, and in particular relates to an artificial Pseudomonas aeruginosa phage lyase (Endolysin or wall-lysing enzyme) and its application as a bactericidal active component in food biofilms. Background technique [0002] Biofilm (Bacterial biofilm) refers to bacteria in order to adapt to the environment, adhere to the interface of different materials, secrete a large amount of heterogeneous extracellular matrix such as exopolysaccharide, protein and nucleic acid, and wrap the bacteria itself in it. Bacteria aggregate membrane-like objects. Biofilms provide bacteria with a protective lifestyle, and their formation facilitates continued microbial colonization, resistance to clearance by the host immune system, increased resistance to antibiotics, and exchange of genetic material. The biofilm adheres to the contact surface, and bacteria are embedded in the polymer protective layer, which makes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N15/56C12N15/70C12N1/21A23L3/3571
CPCA23L3/3571A23V2002/00C12N9/2462C12N15/70C12Y302/01017A23V2200/10
Inventor 杨洪江荆兆元何洋张甜
Owner TIANJIN UNIV OF SCI & TECH
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