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

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

Example Embodiment

[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 into 15 mL of LB liquid medium, and cultured with shaking at 36°C for 6-8 hours. Pick a single Salmonella phage SLMP1 plaque, inoculate it in 15mL of the logarithmic growth phase host bacteria culture medium, shake and culture at 36°C for 4-6h, then centrifuge the lysate at 8000rpm for 10min, and filter the supernatant with a 0.22μm filter. , which is the pure phage culture medium. The Salmonella cultured overnight was transferred to 400 mL of liquid LB medium, the inoculum amount was 1%, cultured to the logarithmic growth phase, 10 mL of phage pure culture solution was added, and the culture was shaken until the host bacteria lysed and became clear. DNase I and RNase A were added to a final concentration of 1 mg / L and incubated at room temperature for 30 min. ...

Example Embodiment

[0035] Example 2. Functional annotation of the lyase gene lysSP1

[0036] The extracted phage genomic DNA is sent to a bio-company for whole genome sequencing, the genome is predicted by RAST tool, the ORF sequence predicted by the sample is converted into a protein sequence by the transeq program in EMBOSS, and the protein sequence is compared with public data. , the amino acid sequence (SEQ ID No.1) encoded by the gene (SEQ ID No.2) was found to be highly similar to the lyases of several Salmonella phages through homology analysis. At present, there are no reports on the research and application of these highly similar lyases.

Example Embodiment

[0037] Embodiment 3, the construction of recombinant plasmid

[0038] 1. Amplification of the lyase gene lysSP1: Design primers according to the sequence of lysSP1 (SEQ ID No. 2), add double restriction enzyme 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 electrophoresis was performed in 1.5% agarose to identify the size of the amplified fragment. At the same time, the amplified product was sent to a biological company for sequencing, and the amplified fragment was determined as the target fragment.

[0039] 2. TA cloning of the lyase gene lysSP1: The PCR product was recovered with a gel recovery kit, mixed with the pMD18-T cloning vector, and transferred into 100 μL of E. coli DH5α competent cells, and the mixture was placed on ice for 30 min at 42°C Heat shock for 45 s, then place on ice for 1 min, add 890 μL LB li...

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PUM

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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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