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Lysosome and holin composition for resisting salmonella phage expression, preparation method thereof and application of lysosome and holin composition

A Salmonella and perforin technology, applied in the field of bioengineering, can solve the problem of less types of lysozyme and perforin, and achieve the effects of low cost, simple operation and good bacteriostatic effect

Active Publication Date: 2021-01-22
SOUTHWEST UNIV
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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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  • Lysosome and holin composition for resisting salmonella phage expression, preparation method thereof and application of lysosome and holin composition
  • Lysosome and holin composition for resisting salmonella phage expression, preparation method thereof and application of lysosome and holin composition
  • Lysosome and holin composition for resisting salmonella phage expression, preparation method thereof and application of lysosome and holin composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation and Purification of Salmonella 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 Salmonella;

[0038]Shake culture overnight at 37°C, centrifuge the overnight liquid, filter the supernatant with a filterless membrane, mix it with Salmonella liquid and TSB containing 0.7% agar, pour it into a TSA plate, and culture it at 37°C. Phage isolation;

[0039] Purify phage plaques until a single plaque morphology appears on the plate; add 3mL SM buffer (100mM NaCl, 8mM MgSO 4 ·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 Salmonella bacteria in the logarithmic phase to the supernatant, culture at 37°C, 120rpm until the liquid is comple...

Embodiment 2

[0044] Identification of bacteriophage SM-p 2, acquisition of endolysin Lys 2 and perforin Hol 2 gene fragments

[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 phage SM-p 2 as a template, the endolys...

Embodiment 3

[0063] Cloning, expression and purification of endolysin Lys 2

[0064] The endolysin Lys 2 gene fragment obtained in Example 2 was connected to the pEasy-Blunt E1 vector for blunt-end cloning; the pEASY-Lys 2 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 the LB liquid medium, cultivate overnight at 37°C with 200rpm shaking; inoculate the overnight cultured positive clone strain 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;

[0065] Toxic effect of endolysin Lys 2 expression on Escherichia coli BL21(DE3), add IPTG with a final concentration of 1mmol / L, shake culture at 37°C, 200rmp, and take bacterial liquid every (20min) during the induction ...

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Abstract

The invention provides a lysosome and holin composition for resisting salmonella phage expression, a preparation method thereof and application of the lysosome and holin composition, and belongs to the technical field of bioengineering. The amino acid sequence of lysosome is as shown in SEQ ID NO:1, and the amino acid sequence of holin is as shown in SEQ ID NO:2. The lysosome Lys 2 and the holin Hol 2 for resisting salmonella phage expression have broad-spectrum bactericidal activity, have a relatively good antibacterial effect on various Gram-positive and Gram-negative bacteria, and have an obvious killing effect on salmonellae, and the lysosome Lys 2 has an N-terminal enzyme activity structural domain, and can split beta-1,4-glucosidic bonds, and the holin Hol 2 has a conservative structural domain, can destroy cells, and transfers information to control the lysis time of cells; and the lysosome Lys 2 and the holin Hol 2 can be used for preparing drugs for preventing, inhibiting or treating cell infections, and have popularization and application value in the field 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 Salmonella phage expression, a preparation method and application thereof. Background technique [0002] Salmonella is a common foodborne pathogen considered a major cause of gastrointestinal illness, predisposed to salmonellosis, has a high zoonotic potential, and causes serious and potentially fatal disease in animals and humans Infect. Most human Salmonella infections are usually associated with ingestion of the bacteria from contaminated food of animal origin, including pork, beef, lamb, and poultry. The incidence of drug-resistant Salmonella infections is increasing due to inappropriate use of antibiotics. Drug-resistant Salmonella is included in the World Health Organization (WHO) list of priority pathogens. Globally, drug-resistant Salmonella have been isolated from different food products of anima...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70A61K38/47A61K38/16A61P31/04
CPCC12N15/70C07K14/005C12N9/2434C12Y302/01092A61K38/47A61K38/162A61P31/04C12N2795/10122A61K2300/00Y02A50/30
Inventor 石慧解天慧
Owner SOUTHWEST UNIV
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