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Lyase of endo salmonella bacteriophage as well as coding gene, preparation method and application of lyase

A bacteriophage lyase, Salmonella technology, applied in the field of lyase of endometrial Salmonella phage, can solve the problems of environmental residues, drug-resistant bacteria and the like

Pending Publication Date: 2022-04-08
GUANGDONG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to control Salmonella infection, antibiotics are the most effective method, but the abuse of antibiotics will lead to environmental residues and the emergence of drug-resistant bacteria, lysin secreted by phage is one of the effective methods to solve bacterial resistance

Method used

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  • Lyase of endo salmonella bacteriophage as well as coding gene, preparation method and application of lyase
  • Lyase of endo salmonella bacteriophage as well as coding gene, preparation method and application of lyase
  • Lyase of endo salmonella bacteriophage as well as coding gene, preparation method and application of lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Bioinformatics analysis of lyase L17.

[0034]In the previous work of the present invention, a strain of Salmonella cleavage phage PSM6 has been isolated and identified, and sequenced. (Huang Jingxiao, Shang Junkang, Chen Huimin, et al. Biological characteristics and genome analysis of a virulent Salmonella phage [J]. Biotechnology Bulletin, 37(6):11.). Bioinformatics analysis was performed on one of the lyases, L17. Use EMBOSSTranseq (https: / / www.ebi.ac.uk / Tools / st / emboss_transeq / ) to translate the nucleotide sequence of L17 into protein; use Interpro and Pfam tools to predict and analyze the conserved domain of L17 protein sequence, and use SignalP Tools to analyze signal peptide; TMHMM to analyze transmembrane structure; use psipred and swiss-model to analyze secondary and tertiary structure; use expasy's protparam tool to calculate the isoelectric point and molecular mass of the enzyme to be expressed, Nanjing Detai Biological Mirror Website Analyze rest...

Embodiment 2

[0035] Example 2 Expression and purification of lyase L17.

[0036] L17 gene amplification and construction of recombinant plasmid: According to the L17 gene sequence, use PrimerPremier to design specific primers. The L17 gene was amplified by PCR using the genomic nucleic acid of phage PSM6 as a template. The amplified product and the pCOLDⅡ plasmid were digested with the same restriction endonuclease, and then enzyme-linked. The enzyme-linked product was transformed into competent cells of Escherichia coli TOP10 strain. Recombinants were screened with ampicillin and verified by sequencing. Finally, the recombinant expression plasmid vector pCOLDⅡ-L17 was constructed.

[0037] Transform the pCOLDⅡ-L17 vector into Escherichia coli BL21(DE)3Plyss competent cells: Take 1 μL of the extracted vector pCOLDⅡ-L17 and add it to 100 μL of cloud-like competent bacteria, ice bath for 20 minutes; heat shock at 42°C for 90 seconds, and re- Keep transposed on ice for 5 minutes, add 600 ...

Embodiment 3

[0040] Example 3 The CCK8 method was used to measure the cytotoxic activity of L17.

[0041] Recovery culture of RAW 264.7 cells: Use tweezers to take out the RAW 264.7 cell cryopreservation tube on ice, boil water in the induction cooker and preheat it to 37-40°C, put the cells in warm water and shake gently until the ice cubes in the cryopreservation tube melt. Cells were moved to a biosafety cabinet in a cell room. Take 10mL DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) in a 15mL centrifuge tube, centrifuge at 800rpm for 4min, discard the supernatant, continue to gently pipette the cells in the medium to suspend, and repeat the centrifugation. Add 2 mL of culture medium and gently pipette the cells to suspend them, then move them to a petri dish, continue to add 4 mL of culture media to the petri dish, move to an incubator containing 5% CO2, and culture at 37°C.

[0042] The cytotoxic activity of L17 was measured by CCK8 method; 100 μL of revived Raw 264 ...

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Abstract

The invention belongs to the field of microbial genetic engineering, and particularly relates to endosalmonella lyase derived from salmonella, a coding gene of the endosalmonella lyase and a preparation method and application of expression protein of the endosalmonella lyase. The salmonella lyase L17 is derived from a salmonella strain, and the theoretical isoelectric point (PI) and the molecular weight (mw) of the incision salmonella lyase are 9.08 and 17107.69 respectively. And no signal peptide or transmembrane structure is contained. The gene of the novel salmonella lyase is cloned to an escherichia coli expression vector to obtain an escherichia coli recombinant strain capable of heterologously expressing the enzyme, and the salmonella lyase L17 prepared by heterologously expressing the strain can efficiently degrade salmonella at low temperature or normal temperature.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and in particular relates to a lyase for endocutting Salmonella phage, its coding gene, its preparation method and application. Background technique [0002] Salmonella can cause various syndromes such as gastroenteritis, typhoid fever, sepsis and extraintestinal focal infection, and can occur in the harvest, production, processing, preservation and other processes from farm to consumer in the food chain. In order to control Salmonella infection, antibiotics are the most effective method, but the abuse of antibiotics will lead to environmental residues and the emergence of drug-resistant bacteria, lysin secreted by phage is one of the effective methods to solve bacterial drug resistance. [0003] Lyase is a hydrolase that targets and degrades bacterial cell wall peptidoglycan synthesized after phage infects host bacteria and assembles mature progeny phage. It forms holin-lyase cleavag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70A01N63/50A01P1/00A23L3/3571A23L5/20A23K30/00A61K38/51A61P31/04C12R1/19
CPCY02A50/30
Inventor 林伯坤梁文锐姚婉议王雨露朱慧英尚俊康陈嘉淳陈碧莹黄景晓
Owner GUANGDONG MEDICAL UNIV
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