Genetically engineered lysin with function of killing staphylococcus, as well as preparation method and application thereof

A staphylococcus and genetic engineering technology, applied in the field of bioengineering, can solve the problems of few lytic enzymes and staphylococcus no lytic activity, and achieve the effect of wide cleavage spectrum and strong cleavage activity

Inactive Publication Date: 2020-01-14
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many staphylococcal lyases have been reported, but there are few lyases with strong lytic activity and broad host spectrum across species
Our laboratory has previously expressed Staphylococcus aureus phage lysing enzyme LysGH15 and carried out in vitro and in vivo activity assays. All cocci have high lytic activity and broad host spectrum, but no lytic activity to other staphylococci

Method used

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  • Genetically engineered lysin with function of killing staphylococcus, as well as preparation method and application thereof
  • Genetically engineered lysin with function of killing staphylococcus, as well as preparation method and application thereof
  • Genetically engineered lysin with function of killing staphylococcus, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Expression and purification of lyase

[0028] Amplified lyase LysGH15 (+) The primers are: upstream 5'-CCGCTCGAGATGGCTAAGACT CAAGCAGAA-3', downstream 5'-CGGGATCCCTATTTGAATACTCCCCAGGCAA-3'. The amplified LysGH15 (+) specific restriction site xho I and Bam H I was connected to the pET15b vector to construct the expression vector pET15b-LysGH15 (+) . Transform the constructed vector into Escherichia coli BL21 competent, amplify in 500 mL of fresh LB medium, add 1 / 1000 kanamycin sulfate at the same time, and wait until the bacterial solution OD 600 When the value reached 0.6-0.8, 1 / 1000 IPTG (final concentration 1 mM) was added for induction, the induction temperature was 16°C, and the induction time was 16 h.

[0029] Use Ni-NTA to purify the protein by affinity chromatography: collect the induced bacterial solution (4°C, 10000 × g, 20min), resuspend it with an appropriate amount of Tris-Cl buffer (pH=7.5), and take 80 µL of the whole bacterial sample, and the res...

Embodiment 2

[0037] LyaseLysGH15 (+) Determination of antibacterial ability of solid plate

[0038] Spread Staphylococcus aureus USA300 in the logarithmic growth phase evenly on the TSB solid medium plate in the ultra-clean bench, and after drying, add 10 µL of the purified lyase LysGH15 (+) Drop onto the plate and mark it. In addition, 10 µL of Tris-Cl buffer was dropped on the plate as a control. Place the plate in a 37°C incubator for overnight culture, and observe the formation of the inhibition zone the next day.

[0039] The result is as figure 2 As shown, lucid plaques appear at the markers, indicating that the lyase LysGH15 (+) It has strong lytic activity against Staphylococcus aureus USA300.

Embodiment 3

[0041] LyaseLysGH15 (+) Optimum Action pH Determination

[0042] The same amount of lyase LysGH15 (+) The protein buffer was adjusted to different pH values, that is, the pH value of the 50 mM sodium acetate buffer was adjusted to 4.0-6.0, and the Tris-Cl buffer was adjusted to 7.0-9.0. Then the lyase LysGH15 under different pH conditions (+) With the same amount of Staphylococcus aureus USA300 (OD 600 =0.6) to mix thoroughly, place in a 37°C water bath for incubation, and after 30 minutes, apply each reaction solution on the plate, measure the number of colonies in each reaction solution, and repeat three times. The more the number of colonies decreased, it indicated that LysGH15 was lysed under this pH condition (+) The stronger the cleavage activity of the lyase, the buffer pH value with the strongest cleavage activity of the lyase was defined as the optimal pH value.

[0043] The result is as image 3 shown, indicating that the lyase LysGH15 (+) The optimal pH value...

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Abstract

The invention discloses a genetically engineered lysin with a function of killing staphylococcus and a preparation method. According to the preparation method, a lysin LysGH15 is subjected to genetically engineered modification to obtain a novel bacteriophage lysin LysGH15(+). The bacteriophage lysin LysGH15(+) shows stronger bactericidal activity and wider host spectrum, in comparison with a lysin before modification, can kill staphylococcus aureus, staphylococcus epidermidis, staphylococcus haemolyticus, staphylococcus hominis, staphylococcus saprophyticus, staphylococcus cohnii, fermented staphylococcus xylosus and the like, provides a potential novel medicine to prevention and treatment of diseases caused by staphylococcal infection, and has a good application value.

Description

technical field [0001] The invention discloses a genetically engineered lyase for killing staphylococcus and a preparation method thereof, which is a genetically engineered phage lyase LysGH15 (+) , the lyase has stronger bactericidal activity and wider host spectrum for Staphylococcus compared with the modified lyase, and belongs to the technical field of bioengineering. Background technique [0002] Staphylococci can be divided into coagulase-negative staphylococci and coagulase-positive staphylococci. Staphylococcus aureus ( Staphylococcus aureus ) belongs to coagulase-positive staphylococcus and is an important zoonotic pathogen, which can cause skin abscess, wound infection, endocarditis, osteomyelitis, pneumonia and toxic shock syndrome and other local and systemic infectious diseases . Coagulase-negative staphylococci include Staphylococcus epidermidis ( S. epidermidis ), Staphylococcus saprophyticum ( S. saprophyticus ), Staphylococcus hominis ( S. hominis )...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70A61K38/51A61P31/04
CPCC12N9/88C12N15/70A61P31/04A61K38/00
Inventor 韩文瑜顾敬敏冀亚路郭志敏程梦珺张蕾张玉凤夏翡翡王彬肖峰
Owner JILIN UNIV
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