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Endolytic enzyme and perforin composition for resisting colibacteriophage expression as well as preparation method and application thereof

A technology of Escherichia coli and perforin, applied in the field of bioengineering, can solve the problems of few types of lysozyme and perforin, and achieve low cost, good antibacterial effect, and broad-spectrum bactericidal activity

Active Publication Date: 2021-02-02
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few types of lysozymes and perforins in existing phages

Method used

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  • Endolytic enzyme and perforin composition for resisting colibacteriophage expression as well as preparation method and application thereof
  • Endolytic enzyme and perforin composition for resisting colibacteriophage expression as well as preparation method and application thereof
  • Endolytic enzyme and perforin composition for resisting colibacteriophage expression as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation and Purification of Escherichia coli Phage

[0037] Sewage samples were taken from the river in Chongqing City. After the water samples were centrifuged, CaCl with a final concentration of 1.25mmol / L was added. 2 , take the supernatant and filter it with filter paper, filter it into a sterile container with a 0.22 μm filter membrane, add 2×LB and Escherichia coli O157:H7;

[0038] Shake culture overnight at 37°C, centrifuge the overnight liquid, filter the supernatant with no filter membrane, mix it with Escherichia coli O157:H7 bacteria liquid and TSB containing 0.7% agar, and pour it into a TSA plate, 37 Cultivate at ℃ for phage isolation;

[0039] Purify phage plaques until a single plaque morphology appears on the plate; add 3mL SM buffer (100mM NaCl, 8mM MgSO4 ·7H 2 0, 50mM Tris-HCl, pH=7.5), collect the liquid and the top agar, and centrifuge at 9000×g for 15min;

[0040] Add Escherichia coli O157:H7 bacteria liquid in logarithmic phase to the super...

Embodiment 2

[0044] Identification of bacteriophage EC-p 9, acquisition of endolysin Lys 9 and perforin Hol 9 genes

[0045] Extract the phage DNA with a kit, use the phage DNA as a template, and carry out PCR amplification with random primers (10-mer RAPD) shown in the decamer oligonucleotide sequence SEQ ID NO:5~SEQ ID NO:12, at 94°C Pre-denaturation for 4 minutes; denaturation at 94°C for 1 minute, renaturation at 34°C for 1 minute, extension at 72°C for 2 minutes, 45 cycles, extension at 72°C for 10 minutes;

[0046] According to the amplification results, select primers with few and bright PCR product bands to amplify again, and the PCR products are gel recovered, and the gel recovered products are subjected to TA cloning and sequencing;

[0047] Use NCBI to perform similarity comparison to determine the type of phage;

[0048] Determine the endolysin and perforin primers according to the most familiar phage;

[0049] Using the DNA of bacteriophage EC-p 9 as a template, the endolysi...

Embodiment 3

[0062] Cloning, expression and purification of endolysin Lys 9

[0063] The endolysin Lys 9 gene fragment obtained in Example 2 was connected to the pEasy-Blunt E1 vector for blunt-end cloning; the pEASY-Lys 9 recombinant vector was obtained by blue-white screening, bacterial liquid PCR amplification and sequencing; the obtained recombinant vector Transform into Escherichia coli E.coli BL21(DE3) competent cells, spread on TSA plates containing 100ng / mL ampicillin, and culture at 37°C for 16 hours; In LB liquid medium, culture overnight at 37°C with 200rpm shaking; inoculate the positive cloned strains cultivated overnight into 500mL LB liquid medium (100ng / mL ampicillin) at a dilution ratio of 1:100, at 37°C, Shake culture at 200rpm to OD 600 reach 0.6;

[0064] Toxic effect of endolysin Lys 9 expression on Escherichia coli BL21(DE3), adding IPTG with a final concentration of 1mmol / L, shaking culture at 37°C and 200rmp, and taking bacterial liquid every (20min) during the in...

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Abstract

The invention provides an endolytic enzyme and perforin composition for resisting colibacteriophage expression as well as a preparation method and application thereof, and belongs to the technical field of bioengineering. An amino acid sequence of endolytic enzyme is as shown in SEQ ID NO:1, and an amino acid sequence of perforin is as shown in SEQ ID NO:2. The endolytic enzyme Lys 9 and the perforin Hol 9 expressed by bacteriophage have broad-spectrum bactericidal activity, have a relatively good antibacterial effect on various gram-positive bacteria and gram-negative bacteria, and have an obvious killing effect on escherichia coli; the endolytic enzyme Lys 9 has an N-terminal enzyme activity structural domain and can dissociate beta-1,4-glucosidic bonds; the perforin Hol 9 has a conservative structural domain, can destroy cells and transmits information to control the lysis time of the cells; and the composition can be used for preparing medicines for preventing, inhibiting or treating cell infection, and has popularization and application value in the fields of medical treatment and food safety.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an endolysozyme and perforin composition resistant to the expression of Escherichia coli phage, a preparation method and application thereof. Background technique [0002] Escherichia coli O157:H7 is the most typical food-borne pathogen among enterohemorrhagic Escherichia coli, which can produce Shiga toxin and cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and other diseases, and may even cause death. However, the use of antibiotics to control E. coli O157:H7 is controversial because antibiotics may lead to a large release of Shiga toxin. Clinical studies have shown that antibiotic therapy does increase the risk of developing HUS. In addition, the misuse of antibiotics has led to the emergence of a large number of bacterial resistance, and drug-resistant E. coli O157 has increased. [0003] In recent years, bacteriophage as a new type of anti...

Claims

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Application Information

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IPC IPC(8): C12N9/24C07K14/01C12N15/70C12N1/21A61K38/47A61P31/04A61K38/16C12R1/19
CPCC12N9/24C07K14/005C12N15/70A61K38/47A61K38/162A61P31/04C12N2795/10022A61K2300/00Y02A50/30
Inventor 石慧解天慧
Owner SOUTHWEST UNIVERSITY
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