A kind of Escherichia coli lyase and its preparation method and application

A technology of Escherichia coli and lyase, applied in the field of genetic engineering, can solve the problems of narrow phage lysis spectrum and variation, and achieve the effects of no toxic side effects, safe toxic side effects, and high prevention and control effects

Active Publication Date: 2018-06-05
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the lyase is derived from phage, its application is limited due to the narrow lysis spectrum of phage and the problems of variation in the application process.

Method used

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  • A kind of Escherichia coli lyase and its preparation method and application
  • A kind of Escherichia coli lyase and its preparation method and application
  • A kind of Escherichia coli lyase and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning of recombinant chimeric lyase LysBM1 gene, construction of expression vector and expression strain

[0036] (a) Design the chimeric lyase LysBM1 gene according to the structural characteristics of E. coli cell wall and the lyase gene sequence of E. coli virulence phage JS25 isolated by our laboratory, and its nucleotide sequence is shown in Seq ID NO.1

[0037](ATGCARATHTTYAAYATGMGNACNAAYGAYGARGGNGTNYTNGGNYTNMGNYTNACNYTNTAYAARGAYACNMGNGGNTTYTGGACNATHGGNGCNGGNATHTGGACNGTNTGYGCNAAYCCNGTNTGYGCNGGNGCNYTNGAYMGNATGATHGGNMGNAARTGYAAYCCNACNGTNTGYYTNYTNAARGCNMGNAARCARTTYAAYAARAARGTNAAYAARGCNGGNMGNGGNGCNATHGCNGGNAAYGCNWSNGTNAARCCNGTNTAYAARWSNYTNAARGCNGGNYTNGCNWSNGCNYTNGTNAAYATGGCNTTYMGNMGNGCNWSNTTYCCNTAYAAYGTNGCNTTYMGNWSNYTNMGNYTNYTNAARAARCARAARMGNTGGMGNAAYYTNAAYGCNGGNAARGCNTGYMGNAARTGGTAYMGNCARACNCCNAAYMGNGCNAARMGNGTNATHAARACNTGGGCNGGNGGNAARGTNWSNMGNMGNGAYATHGAR)。

[0038] (b) Construction of the recombinant Pichia expression vector of the lyase: according to th...

Embodiment 2

[0041] Example 2 Induced expression and purification of chimeric lyase LysBM1 protein

[0042] Induced expression: pick positive clones and inoculate them in 50ml BMGY medium (yeast extract 1%, tryptone 2%, potassium phosphate buffer (pH6.0) 100mmol / L, YNB 1.34%, biotin (4×10 -5 )% glycerol 1%), 30°C, 250rpm shaking culture until OD600 is between 4-6, take it out, centrifuge at 1500g for 5min, collect yeast cells, and then resuspend in 1 / 5 volume of BMMY medium (yeast extract 1%, tryptone 2%, potassium phosphate buffer (pH6.0) 100mmol / L, YNB 1.34%, biotin (4×10 -5 )%, methanol 3%) to continue culturing in the Erlenmeyer flask, after 24 hours, add methanol every day to a final concentration of 2.5-3%. The culture was continued for 6-10 days, and the supernatant was collected by centrifugation.

[0043] The supernatant of chimeric lyase LysBM1 yeast expression was directly taken for SDS-PAGE, and electrophoresis showed that the target protein was expressed, and the protein amo...

Embodiment 3

[0044] Example 3 Analysis of the bactericidal spectrum of the chimeric lyase LysBM1

[0045] The lyase with a final concentration of 10 μg / ml was selected to act on Escherichia coli ATCC25922 and 22 strains of Escherichia coli isolated clinically, as well as Staphylococcus aureus ATCC25923, Salmonella CMCC (B) 50115, and Pseudomonas aeruginosa ATCC27853 for detection The ability to form an inhibition zone on the plate. As shown in Table 1, the cleavage spectrum of the lyase LysBM1 is much larger than that of the phage V_EcoM_BM1, and it can lyse Escherichia coli in a broad spectrum.

[0046] Table 1 Comparison of cleavage profiles of lyase LysBM1 and phage V_EcoM_BM1

[0047]

[0048] Among them, "-" means no lysing; "+" means incomplete lysing, and 12 hours later, there are colonies growing in the inhibition plaque; "++" means complete lysing.

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PUM

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Abstract

The invention relates to the field of biological engineering, in particular to an Escherichia coli lyase as well as a preparation method and application thereof as an anti-infection drug. The Escherichia coli lyase is characterized in that an amino acid sequence is Seq ID NO.2. Enzymic preparations capable of effectively killing Escherichia coli are developed by utilizing a bioengineering technology. The preparations can be used independently or can be compounded to use, can specifically deactivate multiple Escherichia coli, and a safe and nontoxic enzyme preparation source with no side effect can be provided for controlling the Escherichia coli in garget at present.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an Escherichia coli lyase, a preparation method and application thereof. Background technique [0002] Escherichia coli is an important zoonotic pathogen. It can cause animal diarrhea, hemorrhagic enteritis, and severe cases can lead to death. The Escherichia coli superbacteria produced by the abuse of antibiotics over the years has brought great harm to human health and the livestock and poultry industry, and it is urgent to develop new and safe prevention and control measures. [0003] Phage lyase is considered to be an efficient biological enzyme antibiotic. In the current situation of increasingly serious microbial drug resistance, the application prospect of lyase in the field of animal disease prevention and control is highly concerned by scientists all over the world. Although the lyase is derived from bacteriophage, its application is limited due to the narrow lysis s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/81A61K38/51A61P31/04
CPCA61K38/00C12N9/88C12N15/815Y02A50/30
Inventor 孙利厂王冉张莉莉何涛庞茂达
Owner JIANGSU ACAD OF AGRI SCI
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