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Pseudomonas aeruginosa bacteriophage endolysins, and coding gene and application thereof

A technology of Pseudomonas aeruginosa and bacteriophage, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of no endolysin resistance, no neutralization of antibodies, etc., to improve bactericidal activity, high activity, inhibiting Broad spectrum effect

Inactive Publication Date: 2018-08-17
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a protein macromolecule, endolysin may produce corresponding antibodies to neutralize its bactericidal ability in the body, but some experiments have found that its antibodies have no neutralizing effect on it
Compared with phage endolysin, it is not easy to produce drug resistance. Idelevich et al. found that the chimeric enzyme PRF-119 is resistant to phage-resistant Staphylococcus aureus mutant strains. There is no related report of endolysin resistance.

Method used

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  • Pseudomonas aeruginosa bacteriophage endolysins, and coding gene and application thereof
  • Pseudomonas aeruginosa bacteriophage endolysins, and coding gene and application thereof
  • Pseudomonas aeruginosa bacteriophage endolysins, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Isolation and identification of phage

[0060] 1. Isolation of phage

[0061] Take 1L of sewage before hospital treatment, add CaCl 2 Centrifuge at 5000rpm for 10min at 4°C to 1mmol / L, remove the precipitated particles in the sewage, take the supernatant and filter it with a 0.22μm filter membrane to sterilize; take 20mL of the filtrate and mix it with 20mL 2×LB medium, and use 1% inoculum , inoculate 400 μL of Pseudomonas aeruginosa in the logarithmic growth phase, and enrich and culture at 37° C. for 12 hours. Take 5ml of the above-mentioned bacterial solution and centrifuge at 5000rpm for 10min at 4°C, and filter the supernatant through a 0.22μm filter membrane to obtain the phage stock solution. The obtained phage stock solution was serially diluted 10 times (10 -1 -10 -8 ), respectively take 300 μL of the dilution and mix it with Pseudomonas aeruginosa in the logarithmic growth phase at a ratio of 1:1, incubate at 37°C for 15 minutes, mix with 4mL of...

Embodiment 2

[0064] Embodiment 2: Extraction of phage genome

[0065] 1. Concentration of phage particles

[0066] The overnight culture of Pseudomonas aeruginosa was transferred to 100mL liquid LB medium, the inoculum size was 1%, and the culture was expanded to the logarithmic phase (OD600 about 0.4), and 5mL Pseudomonas aeruginosa phage culture solution was added, 37 The phage lysates were obtained after 4-6 hours of shaking culture at ℃. Add DNase I and RNase A to the lysate to a final concentration of 5 μg / mL, mix well and let stand at 37°C for 1 h. Then add NaCl to a final concentration of 0.1mol / L, mix and dissolve, put in ice bath for 1h, and centrifuge at 12000rpm for 20min. After the supernatant was transferred to another centrifuge tube, PEG8000 was added to a final concentration of 10% (w / v), fully shaken to dissolve, then left at 4°C overnight, centrifuged at 12,000 rpm for 20 min, and the supernatant was discarded. Resuspend the precipitate with 500 μL TM (0.05mol / L Tris-H...

Embodiment 3

[0076] Example 3: Cloning of endolysin gene Lysin-G78, construction of expression vector

[0077] 1. Obtaining the target fragment

[0078] S1 designs a pair of specific primers according to the sequence encoded by the Lysin-G78 (sequence 2) gene, and the primer sequences are as follows:

[0079] Upstream primer: 5'-GC GGATCC ATGATCACCGACAGAGAGTATCAG-3', wherein, the underlined part is the restriction site of BamHI

[0080] Downstream primer: 5'-GC CTCGAG TCAGCCACTAGCTTCAGCATA-3', wherein, the underlined part is the enzyme cleavage site of Xho I

[0081] The reflection system is:

[0082]

[0083] PCR reaction conditions: pre-denaturation at 95°C for 5 min, 30 cycles of amplification (denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1.5 min); extension at 72°C for 10 min, storage at 4°C. PCR amplification products were subjected to 1% agarose gel electrophoresis to observe whether there were specific bands.

[0084] Using the phage ge...

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Abstract

The invention discloses pseudomonas aeruginosa bacteriophage endolysins, and a coding gene and application thereof. The invention provides a protein which is as shown in (a) or (b) as below: (a) the protein which is as shown in a sequence 1 in a sequence table and consists of an amino acid sequence; (b) the protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence which is as shown in the sequence 1 in the sequence table, has a function of cracking gram-negative bacteria and is deviated from the sequence 1 in the sequencetable. The protein provided by the invention can crack a plurality of gram-negative bacteria, such as Escherichia coli, Klebsiella pneumonia, pseudomonas aeruginosa, Burkholderia cepacia and Salmonella typhimurium. The protein and a surfactant EDTA (Ethylene Diamine Tetraacetic Acid) act synergistically to achieve a stronger inhibiting effect on the gram-negative bacteria. A foundation is laid for researching and developing a new antibacterial preparation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Pseudomonas aeruginosa phage endolysin, its coding gene and its application. Background technique [0002] In recent years, many bacteria have developed drug resistance, and even multi-drug resistant bacteria have emerged. Multi-drug resistant bacteria pose a serious threat to people's health. Therefore, it is urgent to find a new type of antibacterial agent to deal with the threat of drug-resistant bacteria. As a bacterial virus, bacteriophage has the advantages of high specificity and no side effects on the human body, and has been valued by scientists. Bacteriophage is a virus that can infect bacteria, fungi, actinomycetes and spirochetes, and its content in nature is about 10 31 , or it can be said that there are about 10 7 Phage / cm 3 . Phages can be divided into lysogenic and lytic phages. The lysogenic phage coexists with the genome combination of its own genome and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/70A61K38/47A61P31/04
CPCA61K38/00A61P31/04C12N9/24C12N15/70Y02A50/30
Inventor 徐永平渠坤丽袁玉玉王丽丽李晓宇
Owner DALIAN UNIV OF TECH
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