Enterococcus faecalis phage lyase and coding gene and application thereof

A technology of Enterococcus faecalis and Enterococcus faecium, applied in lyase, application, genetic engineering and other directions, can solve problems such as health threats and achieve good therapeutic effect

Inactive Publication Date: 2014-03-26
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vancomycin was once considered the last line of defense against drug-resistant bacteria, but with the frequent emergence of vancomycin-resistant Enterococcus faecalis and the rapid sp

Method used

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  • Enterococcus faecalis phage lyase and coding gene and application thereof
  • Enterococcus faecalis phage lyase and coding gene and application thereof
  • Enterococcus faecalis phage lyase and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1, Isolation and identification of Enterococcus faecalis phage IME-EF1

[0070] 1. Isolation of Enterococcus faecalis phage

[0071] Enterococcus faecalis 002 was used as an indicator bacterium to isolate phage from hospital sewage, and the specific steps were as follows:

[0072] (1) Sewage pretreatment: Take the sewage without any disinfection treatment from the sewage treatment station of the 307 Hospital of the Chinese People's Liberation Army, centrifuge it at 10000rpm for 10min, collect the supernatant, filter it with a 0.45um microporous membrane, and collect the filtrate;

[0073] (2) Streak Enterococcus faecalis 002 glycerol bacteria on solid BHI medium and culture overnight at 37°C;

[0074] (3) Pick a single clone and inoculate it in BHI medium, culture it in a shaker at 220rpm, 37°C to logarithmic growth phase, add 100μl of sewage filtrate, shake it at 37°C overnight, and then centrifuge at 10000rpm for 10min, collect the supernatant, And use a 0.4...

Embodiment 2

[0149] Embodiment 2, expression and activity detection of Lysin-EF1 protein

[0150] 1. Construction of recombinant expression vector pColdI-Lysin-EF1

[0151] 1. Acquisition of Lysin-EF1 gene

[0152] Using the genomic DNA of phage IME-EF1 as a template, primer 1 and primer 2 were used for PCR amplification.

[0153] Reaction system: 0.5 μl of genomic DNA of phage IME-EF1, 10 μl of 5×Buffer, 4 μl of dNTPs, 0.5 μl of Pusion enzyme, 2.5 μl of upstream and downstream primers, and make up to 50 μl with deionized water.

[0154] Reaction conditions: pre-denaturation at 98°C for 30s; denaturation at 98°C for 15s, annealing at 55°C for 30s, extension at 72°C for 1min, a total of 35 cycles; final extension at 72°C for 7min.

[0155] Primer 1: 5'-GC GGATCC ATGGTTAAATTAAACG-3' (the underlined part is the recognition site of BamH I, and the following sequence is the 1-16th position of sequence 2);

[0156] Primer 2: 5'-GC TCTAGA CTATACTTTAACTTGT-3' (the underlined part is the rec...

Embodiment 3

[0233] Embodiment 3, Lysin-EF1 protein therapeutic effect analysis

[0234] In this example, a mouse model of abdominal infection with Enterococcus faecalis 002 was established, treated with recombinantly expressed Lysin-EF1 protein, and the therapeutic effect was evaluated by indicators such as mouse mortality and bacterial concentration in blood.

[0235] SPF-grade Balb / C mice: dozens, female, 6-8 weeks old, purchased from the Experimental Animal Center of the Academy of Military Medical Sciences.

[0236] 1. Determination of the minimum lethal dose in mice

[0237] In order to establish a mouse abdominal infection model, the minimum lethal dose (MLD) and minimum lethal concentration (MLC) of Enterococcus faecalis 002 to the same batch of mice were determined in advance, and the specific measurement methods are as follows:

[0238] (1) Bacteria preparation: Inoculate 5ml of Enterococcus faecalis 002 bacteria solution in the logarithmic growth phase into 1L of fresh BHI medi...

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Abstract

The invention discloses enterococcus faecalis phage lyase and a coding gene and application thereof. The enterococcus faecalis phage lyase is a protein of the following (a) or (b): (a) protein composed by an amino acid sequence as shown in a sequence 1 of a sequence table; (b) protein which is derived by the sequence 1, has a function of cracking enterococcus faecalis, and is made by the fact that the amino acid sequence as shown in the sequence 1 is subjected to the replacement and/or deletion and/or addition of one or a few of amino acid residue(s). Experiments prove that the lyase protein can crack a variety of kinds of enterococcus faecalis and can be closely combined with the specificity of sensitive bacteria. In addition, animal experiments prove that the lyase has a better treatment effect on the infection caused by enterococcus faecalis, and timely and effective dosage after infection has a certain meaning for reducing the mortality of mice. According to the invention, a foundation for antibiotic replacement therapy resistant to enterococcus faecalis is laid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an Enterococcus faecalis phage lyase, its coding gene and application. Background technique [0002] Enterococcus faecalis, also known as Streptococcus faecalis, is a gram-positive bacterium. According to the Lancefield serological classification, Streptococcus can be divided into many groups, and Enterococcus faecalis belongs to Group D among them. Group D Streptococcus is further divided into Enterococcus and non-Enterococcus, among which Enterococcus includes Enterococcus faecalis (S.faecalis), Enterococcus faecium (S.faecium) and Enterococcus fortitude (S.durans). In recent years, through DNA-DNA hybridization analysis, it was found that the homology between Enterococcus and Streptococcus was low, so Enterococcus was separated and established as Enterococcus. The enterococci associated with human infectious diseases are E.faecalis and E.faecium, of which E.faecalis accounts for 9...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/63A61K38/51A61P31/04
CPCA61K38/00C12N9/88
Inventor 童贻刚米志强张文惠安小平张志毅黄勇李玉元范航范华昊
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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