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Genetic engineering-modified staphylococcus aureus staphylophage lyase as well as preparation method and application thereof

A phage lysing enzyme and Staphylococcus technology, applied in the field of Staphylococcus aureus phage lysing enzyme and preparation, can solve problems such as difficult to produce resistant bacteria, antibiotic residues in milk, and health hazards

Active Publication Date: 2015-08-26
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The emergence of new antibiotics will lead to more drug-resistant Staphylococcus aureus, which is more difficult to control, thus forming a vicious circle
Second, antibiotic residues in milk caused by the treatment of Staphylococcus aureus infection have brought serious food safety hazards and indirectly endangered human health
More and more in vivo animal experiments at home and abroad have shown that lyase has high bactericidal properties, a wider lytic spectrum than bacteriophage, and it is difficult to produce resistant bacteria, lysing of drug-resistant pathogenic bacteria and other antibacterial agents Efficiency of synergistic effect, and antibodies produced in animals by in vivo use of lyase did not affect the effect of lyase

Method used

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  • Genetic engineering-modified staphylococcus aureus staphylophage lyase as well as preparation method and application thereof
  • Genetic engineering-modified staphylococcus aureus staphylophage lyase as well as preparation method and application thereof
  • Genetic engineering-modified staphylococcus aureus staphylophage lyase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Staphylococcus aureus YZ-12, YZ-15, YZ-16, YZ-19, YZ-34, YZ-42, YZ-49, YZ-56, XG-9, XG-25, SF- 16. SF-22, SF-37, SF-46, SF-54, SF-56, A14, A16, X5-1, X12, M-9, S4 and S6 were all isolated, identified and preserved in this laboratory. Typhimurium Salmonella (HNER178, Salmonella Typhimurium) is also isolated and preserved in this laboratory, and Staphylococcus aureus (ATCC25923), Salmonella enteritidis (ATCC13076, Salmonella Enteritidis) and Escherichia coli (ATCC25922) are all purchased from ATCC. Escherichia coli TransB (DE3) is an expert in the art pET32b(+) is a commonly used plasmid in this field.Example 1: Cloning of lyase LysS1 gene (lysS1) and construction of expression vector.

[0045] a. First design the lyase LysS1 gene, name it the lyase LysS1 gene, and its nucleotide sequence is Seq ID NO.1;

[0046] b. The lyase LysS1 gene was synthesized by Shanghai Sangong and subcloned to obtain a recombinant expression plasmid and named pET32b-LysS1; the next day, trans...

Embodiment 2

[0049] Example 2: Induced expression and purification of lyase LysS1 protein

[0050] Inoculate the recombinant strain TransB (pET32b-LysS1) into LB culture medium containing ampicillin (50 μg / mL), shake overnight at 37°C; the next day, transfer to 100mL LB medium at a ratio of 1:100, and inoculate at 37°C Shake culture to OD 600 When the value is about 0.5, add IPTG to a final concentration of 1.0mmol / L, and induce at 26°C for 20h. Collect the bacteria, disrupt the cells by ultrasonic, centrifuge at 10,000 rpm / min at 4°C for 10 min, collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, and analyze the protein expression in the lysed supernatant by SDS-PAGE. The filtered lysed supernatant was purified with a His affinity chromatography nickel column (GE Healthcare, Sweden), specifically according to the instructions of the kit. The obtained protein was named LysS1, and the purified LysS1 product was detoxified (≤0.01 EU / μg endotoxin) through a de...

Embodiment 3

[0052] Embodiment 3: zymogram analysis of lyase LysS1

[0053] Divide the LB (containing 1.2% agarose) plate into several areas, absorb the overnight culture of different host bacteria: Staphylococcus aureus YZ-12, YZ-15, YZ-16, YZ-19, YZ-34, YZ- 42, YZ-49, YZ-56, XG-9, XG-25, SF-16, SF-22, SF-37, SF-46, SF-54, SF-56, A14, A16, X5-1, X12, M-9, S4, S6, all of the above are isolated, identified and preserved in this laboratory and Staphylococcus aureus (ATCC25923), 0.1mL is dropped on the center of the TSB plate, spread the bacterial solution evenly, and incubate at 37°C for 1 hour; At the same time, 0.1 mL of Salmonella typhimurium (HNER178, Salmonella Typhimurium (separated and preserved in our laboratory), Salmonella enteritidis (ATCC13076, Salmonella Enteritidis) and Escherichia coli (ATCC25922) were dropped on the TSB plate and spread evenly; The vector plasmid (pET32b) induced expression product and the buffer solution where the enzyme was located were 10 μL each, and wer...

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Abstract

The invention provides a genetic engineering-modified staphylococcus aureus staphylophage lyase, the amino acid sequence of which is shown in SEQ ID NO:1. The invention also discloses a preparation method of the staphylococcus aureus staphylophage lyase, comprising the following concrete steps: (1) cloning the amino acid sequence shown in the SEQ ID NO:1 into a prokaryotic expression vector to obtain a recombinant plasmid; (2) transforming the recombinant plasmid obtained in the step (1) into host bacteria to obtain recombinant bacteria; (3) expressing the staphylococcus aureus staphylophage lyase by the recombinant plasmid; and (4) purifying the staphylococcus aureus staphylophage lyase obtained in the step (3). Multiple staphylococcus aureus can be specifically inactivated by independently using the genetic engineering-modified staphylococcus aureus staphylophage lyase or matching the genetic engineering-modified staphylococcus aureus staphylophage lyase with other compounds, thus providing a safe enzymic preparation source without toxic and side effects for staphylococcus aureus infection, particularly staphylococcus aureus caused dairy mastitis in the existing dairy farm control.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a Staphylococcus aureus phage lyase, a preparation method and application thereof. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is an important zoonotic pathogen, which can cause wound infection, endocarditis, osteomyelitis, toxic shock syndrome and other diseases. The incidence of mastitis disease in dairy cows is as high as 35-59%, which has greatly affected the milk production of dairy cows in my country, the quality of milk produced, the economic benefits of dairy farms, and even the international competitiveness and reputation of my country's milk quality. [0003] At present, dairy cow mastitis caused by S. aureus infection is mainly controlled and treated by 1) systemic injection of antibiotics, udder injection or infusion, nipple infiltration, 2) immunization of S. Although antibiotics can suppress the bacterial infection ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A61K38/51A61P31/04
CPCA61K38/00C12N9/88
Inventor 周艳王冉张辉包红朵孙利厂
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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