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Chimeric enzyme ClyQ for degrading staphylococcus biofilm as well as preparation method and application of chimeric enzyme ClyQ

A staphylococcus and biofilm technology, applied in the field of chimeric enzymes, can solve the problems of ICU wards of the medical system, limited research on chimeric enzyme removal of biofilms, and difficulty in removing biofilms, etc., and achieves stable performance and high prevention and control effects.

Pending Publication Date: 2022-07-12
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As we all know, Staphylococcus aureus is very easy to form a biofilm that is difficult to remove on the surface of medical equipment, which brings great troubles to the medical system, especially the ICU ward
At present, the research on the use of natural phage lytic enzymes to remove biofilms has been very rich, but the research on the use of chimeric enzymes to remove biofilms is still very limited.

Method used

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  • Chimeric enzyme ClyQ for degrading staphylococcus biofilm as well as preparation method and application of chimeric enzyme ClyQ
  • Chimeric enzyme ClyQ for degrading staphylococcus biofilm as well as preparation method and application of chimeric enzyme ClyQ
  • Chimeric enzyme ClyQ for degrading staphylococcus biofilm as well as preparation method and application of chimeric enzyme ClyQ

Examples

Experimental program
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preparation example Construction

[0034] The present invention also provides a method for preparing the above-mentioned chimeric enzyme ClyQ, comprising the following steps: (1) constructing a recombinant expression vector comprising the encoding gene of the chimeric enzyme ClyQ; the basic vector of the recombinant expression vector includes a prokaryotic expression vector ;

[0035] (2) transforming the recombinant expression vector into prokaryotic cells to obtain an engineered bacterium expressing the chimeric enzyme ClyQ;

[0036] (3) Picking a single colony of the engineered bacteria for induction culture, centrifuging and breaking the cells, and collecting the broken supernatant, the supernatant contains the chimeric enzyme ClyQ.

[0037] The present invention constructs a recombinant expression vector comprising the encoding gene of the chimeric enzyme ClyQ; the basic vector of the recombinant expression vector includes a prokaryotic expression vector. In the present invention, the product constructed ...

Embodiment 1

[0049] Expression and purification of chimeric enzyme ClyQ

[0050] 1.1 Construction of recombinant expression vector expressing chimeric enzyme ClyQ

[0051] 1.1.1 Preparation of the catalytic domain CHAP of LysGH15 and the cell wall binding domain SH3b of PlyV12

[0052]The complete gene fragments of lyase LysGH15 and PlyV12 were synthesized (Beijing Qingke Biotechnology Co., Ltd.), and the synthesized sequences were respectively connected to the pET28a vector. Using LysGH15 gene as a template, using LysGH15CHAP-F (SEQ ID NO.3) and LysGH15CHAP-R (SEQ ID NO.4) as primers, the catalytic domain CHAP of LysGH15 was obtained by amplification. (SEQ ID NO.5) and PlyV12SH3b-R (SEQ ID NO.6) were used as primers to amplify the binding domain SH3b of PlyV12.

[0053] The steps for amplifying the above fragments are as follows:

[0054] Amplification system: 2 μL of template, 2.5 μL of each 10 μM primer, 25 μL of Primerstar 2x Mix, 50 μL of total system, with ddH 2 O make up 50 μL. ...

Embodiment 2

[0066] Broad-spectrum results of in vitro lysis of Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Staphylococcus squirrels, Staphylococcus vatus, Staphylococcus koyeli, and Staphylococcus mimicus by the chimeric enzyme ClyQ.

[0067] A variety of different Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus epidermidis, Staphylococcus squirrels, Staphylococcus var., Staphylococcus kolokii and Staphylococcus mimicus were grown to log phase (OD). 600 = 0.6), low temperature centrifugation (4°C, 6000rpm) to collect the precipitate, washed twice with PBS and then resuspended with PBS to obtain bacterial solutions of different strains. Take 100 μL of the chimeric enzyme ClyQ in Example 1 and mix it with 100 μL of the above bacterial solution, so that the final concentration of the chimeric enzyme ClyQ is 50 μg / ml, and the system OD 600 =0.6~0.8.

[0068] At the same time, the mixture of an equal amount of PBS and the above bacterial so...

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Abstract

The invention provides chimeric enzyme ClyQ for degrading a staphylococcus biofilm as well as a preparation method and application of the chimeric enzyme ClyQ, and relates to the technical field of chimeric enzymes. The chimeric enzyme ClyQ structurally comprises a catalytic domain CHAP of lyase LysGH15 and a cell wall binding domain SH3b of lyase PlyV12, can be used for killing various staphylococcus, has a good removal effect on a biofilm of drug-resistant staphylococcus aureus, and provides a material source for industrial production of drugs for removing staphylococcus biofilms.

Description

technical field [0001] The invention belongs to the technical field of chimeric enzymes, and in particular relates to a chimeric enzyme ClyQ for degrading Staphylococcus biofilm and a preparation method and application thereof. Background technique [0002] Staphylococcus aureus is a common gram-positive bacterium and an important zoonotic pathogen, widely present in air, water, dust and human and animal excrement. In addition, Staphylococcus aureus can cause a variety of diseases in humans and animals, including wound infection, cow mastitis, pseudomembranous colitis, sepsis and sepsis, etc., which seriously threaten the safety of human and animal life. In recent years, due to the extensive use of antibiotics, drug-resistant "superbugs", such as Methicillin-resistant S. aureus (MRSA), continue to emerge. Vancomycin has always been the drug of choice for the clinical treatment of gram-positive bacterial infections, and was once called the "last bottom line" of clinical anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C07K19/00C12N15/70A01N63/50A01P1/00C12R1/19
CPCC12N9/88C12N15/70A01N63/50C07K2319/00A01N63/20Y02A50/30
Inventor 钱平李鑫鑫李祥敏段小超张奋强王爽
Owner HUAZHONG AGRI UNIV
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