Salmonella broad-spectrum lyase with in-vitro lytic activity and application thereof

A Salmonella, lyase technology, applied in the field of broad-spectrum lyase and its antibacterial application, can solve the problems of lack of high-efficiency lyase for Gram-negative bacteria, infection, unsuitable lyase, etc., to facilitate fermentation and large-scale production, Wide cracking spectrum and high bactericidal efficiency

Active Publication Date: 2020-07-31
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, compared with Gram-positive bacteria, because the outer membrane of Gram-negative bacteria can block the effective binding of lyases to peptidoglycan targets, lyases cannot lyse Gram-negative bacteria when they are added from the outside. Lack of highly efficient lyases against Gram-negative bacteria
In order to solve the problem of Salmonella infection, researchers have expressed Lys68, LysSTP4, SPN1S, gp146, Gp110, Lys394, PsP3gp10, LysT144 and other lyases targeting Sal...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Salmonella broad-spectrum lyase with in-vitro lytic activity and application thereof
  • Salmonella broad-spectrum lyase with in-vitro lytic activity and application thereof
  • Salmonella broad-spectrum lyase with in-vitro lytic activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Example 1 Cloning of LysMD19 gene, construction of recombinant expression vector and expression strain

[0029] Our laboratory isolated a large number of Salmonella phages. Through the analysis of the bactericidal activity and host spectrum of these phages, it was found that a Salmonella phage (self-named vB_SenS_MD1) had a broad-spectrum bactericidal activity, and it was speculated that the lyase ( The applicant self-named the lysing enzyme LysMD19, whose amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2) can exert broad-spectrum bactericidal activity, so follow-up research was carried out on the lyase :

[0030] Cloning of LysMD19 gene: PCR conditions are: first 95°C for 5 minutes; then cycle, 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, a total of 30 cycles; finally 72°C for 10 minutes. After the reaction, use 1% agarose gel electrophoresis to detect the fragments. If the target band appears (the target...

Embodiment 2

[0034] Example 2 Induced expression and purification of lyase LysMD19

[0035] Induced expression of the lyase LysMD19: Pick a single colony of the recombinant expression strain with an inoculation loop, place it in a 250mL Erlenmeyer flask with 25mL of BMGY liquid medium, and culture it with shaking at 30°C and 220rpm until the OD600 is about 2; Centrifuge at 5000g for 5min, resuspend the bacteria with BMMY liquid medium, make OD600=1.0, place the bacteria solution in a 1L shake flask, shake and culture at 30°C, 220rpm; add to the medium every 12h for analysis Pure grade methanol to a final concentration of 1.0%; take samples at 24h, 48h, 72h and other time points, collect the supernatant, and filter it with a 0.22μm filter to analyze the expression of the target protein in the supernatant of the yeast expression liquid . The results showed that the expression level of the recombinant expression strain reached the highest value after induction for 48 hours, such as figure ...

Embodiment 3

[0037] Cleavage profile analysis of embodiment 3 lyase LysMD19

[0038] In the test, 20 strains of Salmonella with different serotypes were selected as the test objects, 10 of which were standard control strains, and the other 10 were clinical isolates. The specific operation was as follows: Take 100 μL of Salmonella cultured to the logarithmic phase respectively, and drop them in TSB In the center of the solid medium, use a coating stick to spread them into a uniform lawn. Then take 10 μL of lysing enzyme LysMD19 (concentration: 1 μM) and drop it on the surface of the bacterial lawn. After the droplet is dry, place it upside down at 37°C and incubate for 12 hours. Observe the results. The inhibition zone formed by LysMD19 is as follows: image 3 as shown, image 3 Among them, LysMD17, LysMD18, and LysMD19 are self-named phage lyases expressed by the applicant. The scanning electron microscope observation results of Salmonella typhimurium ATCC13311 treated with lyase LysMD19...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to salmonella broad-spectrum lyase with in-vitro lytic activity and application thereof, and belongs to the technical field of biology. The lyase is named as LysMD19, an amino acid sequence of the lyase is shown as SEQ ID No. 1, and a nucleotide sequence of the lyase is shown as SEQ ID No. 2. The lyase can directly and efficiently lyse salmonella in-vitro under the conditionof no cell permeation reagents such as ETDA, the lysing effect is achieved on salmonella enteritidis, salmonella typhimurium, salmonella pullorum and other salmonella of different serotypes, good temperature stability and high acid and alkali resistance are achieved, the lyase can be used for preventing and treating salmonella infection, and also can be used for killing salmonella in multiple links such as source farms, processing, packaging and transportation to market selling, the safety 'from farms to dining tables' is ensured, and good application prospects and values are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a broad-spectrum lyase with in vitro lytic activity against Salmonella and its antibacterial application. Background technique [0002] Salmonella is a Gram-negative bacillus, which is not only a serious pathogen in livestock and poultry farming, but also an important zoonotic pathogen that causes food poisoning. Among all kinds of bacterial food poisoning worldwide, food poisoning caused by Salmonella often ranks first. Based on the statistical data from 2006 to 2010, Salmonella is the main pathogenic bacteria that caused outbreaks of bacterial foodborne diseases in my country ( 70%-80%), wherein the serotypes that play the main pathogenic role are Salmonella enteritidis and Salmonella typhimurium, and among the foods that cause Salmonella poisoning, meat, eggs, milk and other animal products account for about 90%. Therefore, to solve the public health problem of Salmone...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N9/88C12N15/60A61K38/51A61P31/04A23K20/189
CPCC12N9/88A61K38/51A61P31/04A23K20/189C12Y402/02002Y02A50/30
Inventor 庞茂达王冉孙利厂包红朵张辉
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap