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Staphylococcus aureus bacteriophage lyase as well as preparation method and application thereof

A technology of phage lyase and Staphylococcus, which is applied in the field of Staphylococcus aureus phage lyase and its preparation, and can solve problems such as the lack of separation and purification of Staphylococcus aureus phage lyase

Inactive Publication Date: 2020-10-23
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no effective Staphylococcus aureus phage lyase has been isolated, purified or developed, and used to inhibit and kill Staphylococcus aureus

Method used

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  • Staphylococcus aureus bacteriophage lyase as well as preparation method and application thereof
  • Staphylococcus aureus bacteriophage lyase as well as preparation method and application thereof
  • Staphylococcus aureus bacteriophage lyase as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 Staphylococcus aureus phage lyase gene cloning and vector construction

[0046] 1.1 Strains and vectors

[0047] Staphylococcus aureus phage SA2 was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, with the preservation date being July 27, 2017, and the preservation code is CGMCC No.14331.

[0048] Escherichia coli DH5α competent cells and Escherichia coli BL21 competent cells were purchased from Treasure Bioengineering (Dalian) Co., Ltd.

[0049] The expression vector pCold TF was purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0050] 1.2 Primer design

[0051] According to the whole genome sequence of phage SA2, determine the lysing enzyme LysSA2 gene sequence of phage SA2, and according to the structural domain characteristic of lysing enzyme LysSA2, clone the coding sequence of lysing enzyme LysSA2, lysing enzyme LysSA2-1 and lysing enzyme LysSA2-2 respectively; Wherein The c...

Embodiment 2

[0069] Example 2 Induced Expression and Purification of Recombinant Expression Plasmid Bacteria

[0070] 2.1 Transfer of recombinant expression plasmids into BL21 competent cells

[0071] Take 1 μL of recombinant plasmid and add 100 μL of E. coli BL21 competent cells, mix well, and ice-bath for 30 minutes; heat shock in 42°C water bath for 90 seconds, quickly place on ice for 3 minutes, add 900 μL of LB broth, and incubate at 37°C for 90 minutes; take 100 μL of bacteria The solution was spread on the nutrient agar plate containing Amp and incubated overnight at 37°C.

[0072] 2.2 Induced expression of recombinant expression plasmid bacteria

[0073] Randomly pick a single colony transferred into BL21 competent cells, inoculate it into LB liquid broth containing Amp, culture it with shaking at 37°C until the OD600 is about 0.6-0.8, add IPTG with a final concentration of 0.1mM, and shake overnight at 16°C to induce expression. The recombinant proteins were named lyases LysSA2,...

Embodiment 3

[0077] Embodiment 3 lyase LysSA2, LysSA2-1 and LysSA2-2 in vitro antibacterial activity assay and antibacterial spectrum detection

[0078] 3.1 Determination of antibacterial activity of lyases LysSA2, LysSA2-1 and LysSA2-2 in vitro

[0079] Staphylococcus aureus was cultured to the logarithmic phase in LB liquid medium. After the bacterial liquid was centrifuged, the pellet was washed twice with PBS, and then the bacterial cells were resuspended with PBS and the absorbance OD600 was adjusted to about 0.65. In a 96-well plate, mix 100 μL of the resuspended bacteria with 100 μL of recombinant proteins LysSA2, LysSA2-1 and LysSA2-2 at a concentration of 500 μg / mL, and use the induced expression of the empty plasmid pCold TF protein as a control, 3 in each group In parallel, culture in a 37°C incubator. The OD600 value was measured at intervals of 30 min after the culture started, and the curve of OD600 changing with time was drawn.

[0080]The results showed that the OD600 val...

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Abstract

The invention discloses a staphylococcus aureus bacteriophage lyase as well as a preparation method and application thereof, and relates to the technical field of bioengineering. The invention provides a staphylococcus aureus bacteriophage lyase LysSA2, and the amino acid sequence of the LysSA2 is Seq ID NO.1; and the invention provides an encoding gene of the staphylococcus aureus bacteriophage lyase LysSA2, and the nucleotide sequence of the encoding gene is Seq ID NO.2. The invention also provides a preparation method of the staphylococcus aureus bacteriophage lyase LysSA2. The method clones staphylococcus aureus bacteriophage SA2 to obtain the bacteriophage lyase LysSA2, and carries out induced expression on soluble lyase by adopting a prokaryotic expression mode. The bacteriophage lyase LysSA2 not only has a relatively strong bacteriostatic effect on staphylococcus, but also has a broad-spectrum bacteriostatic effect on staphylococcus from different sources, and lays a foundationfor treating and inhibiting staphylococcus diseases.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Staphylococcus aureus phage lyase and its preparation method and application. Background technique [0002] Staphylococcus aureus is a Gram-positive bacterium, mostly pathogenic, which can cause local suppurative infection. The pathogenicity of Staphylococcus aureus mainly depends on the toxins and invasive enzymes it produces, such as: hemolytic toxin, enterotoxin, deoxyribonuclease, plasma coagulation enzyme, etc. [0003] Because Staphylococcus aureus widely exists in nature and has strong pathogenicity, it is extremely harmful to animal husbandry. A variety of poultry are sensitive to Staphylococcus aureus, umbilical cord infection is more common in chicks, and staphylococcal arthritis and sepsis often occur in adult chickens and broiler breeders. Staphylococcus aureus infection can cause acute, subacute or chronic mastitis, necrotizing staphylococcal dermatitis an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12N15/11C12Q1/527A61K38/51A61P31/04C12R1/19
CPCA61K38/51A61P31/04C12N9/88C12N15/70C12Q1/527
Inventor 潘强任慧英王佳孙虎芝
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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