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Broad-spectrum lyase derived from salmonella bacteriophage and antibacterial application thereof

A bacteriophage lyase and salmonella technology, which is applied in the field of broad-spectrum lyase and its antibacterial application, can solve the problem of inability to kill gram-positive bacteria and the like, and achieve the effect of significant bactericidal effect

Active Publication Date: 2018-09-04
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cloning, expression and isolation of the lyase in Salmonella typhimurium phage SPN1S, it was found that the lyase is stable at pH 7.0-10.5 and temperature 25°C-45°C, EDTA can improve the cleavage activity of the lyase, and the enzyme can kill Escherichia coli , Salmonella, Shigella, Pseudomonas, Cronobacter sakazakii, Vibrio vulnificus and other Gram-negative bacteria, but cannot kill Gram-positive bacteria

Method used

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  • Broad-spectrum lyase derived from salmonella bacteriophage and antibacterial application thereof
  • Broad-spectrum lyase derived from salmonella bacteriophage and antibacterial application thereof
  • Broad-spectrum lyase derived from salmonella bacteriophage and antibacterial application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Extraction of Salmonella phage SLMP1 genome

[0031] 1. Preparation of Salmonella phage SLMP1 particles

[0032] A single colony of Salmonella (ATCC14028) was picked from the solid medium, inoculated in 15 mL of LB liquid medium, and cultured with shaking at 36°C for 6-8h. Pick a single phage plaque of Salmonella phage SLMP1, inoculate it in 15mL logarithmic growth phase host bacterial culture solution, culture it with shaking at 36°C for 4-6h, then centrifuge the lysate at 8000rpm for 10min, and filter the supernatant with a 0.22μm filter membrane , which is the pure culture medium of phage. Transfer the overnight cultured Salmonella to 400mL liquid LB medium with an inoculum size of 1%, cultivate to the logarithmic growth phase, add 10mL phage pure culture solution, and shake the culture until the host bacteria are lysed and become clear. Add DNase I and RNase A to a final concentration of 1 mg / L, and incubate at room temperature for 30 min. Add NaCl (fi...

Embodiment 2

[0035] Example 2, functional annotation of lysase gene lysSP1

[0036] Send the extracted phage genome DNA to the biological company for whole-genome sequencing, use the RAST tool to perform gene prediction on the genome, convert the predicted ORF sequence of the sample into a protein sequence through the transeq program in EMBOSS, and compare the protein sequence with public data , through homology analysis, it was found that the amino acid sequence (SEQ ID No.1) encoded by the gene (SEQ ID No.2) had a high similarity with the lyases of multiple Salmonella phages. At present, there are no research and application reports on these highly similar lyases.

Embodiment 3

[0037] Embodiment 3, the construction of recombinant plasmid

[0038] 1. Amplification of the lysase gene lysSP1: design primers according to the lysSP1 sequence (SEQ ID No.2), add double restriction sites BamH I, Hind III and protective base TTT at the 5' end, upstream primer: 5'- TTTGGATCCatgtcaaaccgaaacatcag-3', downstream primer: 5'-TTTAAGCTTctttgccgcgcgccctac-3'. PCR was used to amplify lysSP1, and electrophoresed in 1.5% agarose to identify the size of the amplified fragment. At the same time, the amplified product was sent to the biological company for sequencing, and the amplified fragment was determined to be the target fragment.

[0039] 2. TA cloning of the lysase gene lysSP1: recover the PCR product with a gel recovery kit, mix it with the pMD18-T cloning vector, transfer it into 100 μL E. coli DH5α competent cells, and place the mixture on ice for 30 minutes at 42°C Heat shock for 45s, then place on ice for 1min, add 890μL LB liquid medium, shake and culture at ...

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Abstract

The invention relates to broad-spectrum lyase derived from salmonella bacteriophage and antibacterial application thereof, and belongs to the technical field of biology. The broad-spectrum lyase is salmonella bacteriophage lyase LysSP1, and the amino acid sequence is SEQ ID NO. 1. After the albumen is subjected to prokaryotic expression and purification, recombination lyase is obtained; the recombination lyase can be cracked into different serological type salmonella, some other gram-negative bacteria and gram-positive bacteria, has a wide lysis spectrum and better bactericidal activity, and can be used for the preparation of antibacterial agents of salmonella and some bacteria.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a broad-spectrum lyase derived from Salmonella phage and its antibacterial application. Background technique [0002] Salmonella (Salmonella) is a kind of Gram-negative bacillus that parasitizes in the intestines of humans and animals, and has similar biochemical reactions and antigenic structures. It is an important food-borne pathogen. Salmonella is mainly transmitted to humans through poultry, meat, milk, eggs, aquatic products, etc. Infection with this bacteria will cause vomiting, diarrhea, fever and other symptoms, and in severe cases, coma or even death. Globally, there are an estimated 93,800,000 gastroenteritis cases and 155,000 deaths each year caused by Salmonella infection, of which about 85% are caused by food transmission. In my country, microbial poisoning incidents caused by Salmonella rank second, but cause the largest number of cases. Therefore, explor...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A61K38/51A61P31/04C12R1/19
CPCA61K38/00A61P31/04C12N9/88C12N15/70
Inventor 江艳华王联珠许东勤姚琳李风铃张媛朱文嘉郭莹莹
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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