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Preparation method of bacteriophage lyase capable of lysing escherichia coli and salmonella

A technology of phage lysing enzyme and Escherichia coli, which is applied in the field of animal biomedical engineering, can solve the problems of difficult prevention and control of Escherichia coli infection, and achieve the effect of not easy denaturation of target protein, easy operation and low cost

Inactive Publication Date: 2017-06-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Carbapenem resistance is a new problem that has emerged recently, mainly caused by plasmid-encoded carbapenemase, and at present, carbapenem resistance mainly appears in E. coli strains in hospitals. Prevention and control poses no small problem

Method used

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  • Preparation method of bacteriophage lyase capable of lysing escherichia coli and salmonella
  • Preparation method of bacteriophage lyase capable of lysing escherichia coli and salmonella
  • Preparation method of bacteriophage lyase capable of lysing escherichia coli and salmonella

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Construction of Escherichia coli BL21 expression strain containing lysin gene

[0039] Include the following steps:

[0040] S1. Construction of recombinant plasmid pET-32a-lysin

[0041] S11. Acquisition of lysin gene: A phage that can lyse both E. coli and Salmonella was isolated from domestic sewage (named ECGD1, see its electron micrograph figure 1 ), extract the genome of the phage after a large number of cultures, and perform high-throughput sequencing and splicing and analysis of the sequencing results.

[0042] Extraction of bacteriophage ECGD1 genome: take 600 μl of phage concentrate after filter sterilization, add DNase I and RNase A to a final concentration of 1 μg / ml, digest overnight at 37°C (to completely remove host bacterial nucleic acid), inactivate at 80°C 15 min (note: this temperature cannot inactivate RNase A); add 24 μl of 0.5 mol / L EDTA (final concentration 20 mmol / L) and 1.5 μl of 20 mg / mL proteinase K (final concentration 50 μg / mL...

Embodiment 2

[0053] Example 2 Activity detection of recombinant protein TrxA-lysin

[0054] Include the following steps:

[0055] S1. Cleavage of chloroform-pretreated DH5α with recombinant protein TrxA-lysin: Inoculate the strain to be tested in LB medium at a ratio of 1%, culture overnight, add 5% chloroform to the bacterial solution, and shake for 15 min , collected by centrifugation, washed twice with deionized water, and stored at -80°C for later use. Before measuring the activity, the bacterial pellet was resuspended with 50 mmol / L Tris-HCl (pH 8.2, containing 0.1% Triton X-100). Add 10 µL of lysing enzyme solution (recombinant protein TrxA-lysin solution) to 90 µL of bacterial suspension, let it stand at room temperature until the lysis effect is obvious, and measure the OD 450 . The experiment was repeated three times, and Tris-HCl buffer was used as a negative control. Since phage ECGD1 can lyse Escherichia coli genetically engineered strain DH5α, DH5α was first used as the te...

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Abstract

Belonging to the field of animal biomedical engineering, the invention specifically discloses a preparation method of a bacteriophage lyase capable of lysing escherichia coli and salmonella. Through primer design, a lyase gene of bacteriophage ECGD1 is successfully amplified by PCR, and is transferred into an expression system to obtain a recombinant strain. IPTG induction is carried out on the recombinant strain to obtain expression, then the strain is lysed to obtain a soluble recombinant lyase of bacteriophage ECGD1, the recombinant lyase can lyse a plurality of escherichia coli and salmonella, i.e. having a wide lysis spectrum. In addition, the synergistic effect result of the recombinant lyase and polymyxin B shows that the existence of the recombinant lyase lowers the minimum inhibitory concentration of polymyxin B, i.e. the two have a synergistic bacteriostatic effect. Identification on the lysis activity of the recombinant lyase and identification on the wide lysis spectrum thereof reveal that the recombinant lyase of the bacteriophage ECGD1 has potential application value in prevention and control of escherichia coli and salmonella infection.

Description

technical field [0001] The invention relates to the technical field of animal biomedical engineering, and more specifically, relates to a preparation method of a phage lyase capable of lysing Escherichia coli and Salmonella. Background technique [0002] Escherichia coli ( Escherichia coli ) is not only a normal resident bacterium in the intestinal tract of humans and animals, but also an important pathogenic bacterium. Escherichia coli can be roughly divided into three categories: intestinal non-pathogenic commensal strains, enteropathogenic strains and extraintestinal pathogenic strains. As a common pathogenic bacteria in humans and animals, it can cause intestinal infection, urinary tract infection, systemic infection and other clinical infections. Extraintestinal pathogenic strains can cause severe extraintestinal infections, especially urinary tract infections, and play an important role in the spread of antibiotic resistance. Among the multidrug-resistant strains of...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70A61K38/51A61K47/64A61P31/04
CPCA61K38/00C07K2319/35C12N9/88C12N15/62C12N15/70Y02A50/30
Inventor 马静云范锦戴麦凯杰冯嘉琪
Owner SOUTH CHINA AGRI UNIV
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