Lyase of bacteriophage and sterilization application

A bacteriophage lyase and bacteriophage technology, applied in the field of bioengineering, can solve the problems of pulmonary edema, irritant toxicity, eye and respiratory tract irritation, etc., and achieve the effects of short action time, broad antibacterial spectrum and good water solubility

Inactive Publication Date: 2016-05-04
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although chlorine dioxide has a good bactericidal effect, it is highly irritating. It mainly causes eye and respiratory tract irritation after contact. Inhalation of high concentrations can cause fatal pulmonary edema.
Patent CN101475879B discloses a bactericidal skin care hand sanitizer with p-chloro-m-xylenol as a bactericide, which also has the disadvantages of strong irritation and toxicity

Method used

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  • Lyase of bacteriophage and sterilization application
  • Lyase of bacteriophage and sterilization application
  • Lyase of bacteriophage and sterilization application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the extraction of phage genome

[0036] (1) Crude phage particles

[0037] (1) Preparation of host bacteria: pick Pseudomonas aeruginosa (Pseudomonasaeruginosa) from solid medium [1] A single colony was inoculated in 5mL LB liquid medium, and cultured with shaking at 37°C for 6-8h.

[0038] (2) Preparation of pure phage culture solution: Pick a single phage plaque, inoculate it in 5 mL logarithmic phase host bacterial culture solution, culture it with shaking at 37°C for 4-6 hours, then centrifuge the lysate at 10000×g for 10 minutes, and the supernatant The solution is the pure culture solution of phage.

[0039] (3) Preparation of crude phage particles: Transfer overnight culture to 100mL liquid LB medium, inoculum size is 1%, amplify and cultivate to logarithmic phase (OD 600About 0.4), add 5 mL of phage pure culture solution, shake culture at 37°C for 6-8 hours to obtain phage lysate. Add DNaseI and RNaseA to the lysate to a final concentration of ...

Embodiment 2

[0049] Embodiment 2, construction of recombinant plasmid

[0050] 1. Obtaining the target fragment

[0051] (1) Primer design

[0052] According to the gene sequence of Pseudomonas aeruginosa bacteriophage K8 lyase, design primers:

[0053] LkesF: 5′-CGCGGATCCATGGCTCTAACTGAGCAAG-3′

[0054] LkesR: 5′-CGCGGATCCTTACTTGAAGGATTGATAGG-3′

[0055] (2) 50μL reaction system:

[0056]

[0057] (3) PCR reaction conditions:

[0058]

[0059] 2. Chemical transformation experiment (construction of pGEM-Teasy recombinant vector)

[0060] (1) Ligate the gp57 gene PCR product with the pGEM-Teasy vector, the connection system is:

[0061] 10μL reaction system:

[0062]

[0063] Mix the reaction system evenly, and connect overnight at 16°C.

[0064] (2) Escherichia coli DH5a chemically competent cell preparation:

[0065] a. Bacterial culture: Pick a single colony of Escherichia coli DH5a in 5 mL of liquid LB medium and culture overnight at 37°C. Transfer the overnight cultur...

Embodiment 3

[0090] Embodiment 3, high expression of lyase in Escherichia coli

[0091] Inoculate the frozen engineered bacteria JK1501 containing positive recombinant plasmids into 20 mL of LB liquid medium (containing ampicillin 100 μg / mL, kanamycin 25 μg / mL), 37 ° C, 220 r / min, shaking culture overnight, the next day, According to the inoculation amount of 1%, the bacterial solution was added to fresh 1 LLB liquid medium (containing ampicillin 100 μg / mL, kanamycin 25 μg / mL), and cultivated to OD at 37 ° C and 180 rpm 600=0.6, add inducer IPTG (to a final concentration of 1 mmol / L). After 4 hours of induction culture, centrifuge at 4000 r / min for 15 minutes to collect the bacteria. Resuspend each 1g of bacteria (wet weight) in 3mL of bacteriostasis buffer (Tris-HCl20mmol / L, NaCl250mmol / L, PMSF1mmol / L, pH7.4), mix well, break the cells by ultrasonic, and centrifuge at 10000× g, centrifuge at 4°C for 15 minutes to remove insoluble cell debris, and filter the supernatant with a 0.22 μm st...

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Abstract

The invention relates to lyase of bacteriophage and sterilization application. The amino acid sequence of the lyase is Seq ID NO.1, the nucleotide sequence of the lyase is Seq ID NO. 2, and hand sanitizer containing the lyase is also provided. The bacteriophage lysine (Endolysin) has the characteristics of high efficiency, a wide spectrum and the like and can be applied in medical treatment and public health, food processing and sanitary products as antibacterial active components. The enzyme preparation can be independently used or used in a compounding mode, bacteria can be specifically inactivated, and a safe enzyme preparation source which is free of toxic and side effects is provided for current bacterial infection prevention and food-derived bacterial contamination prevention.

Description

technical field [0001] The invention belongs to the field of bioengineering, and particularly relates to a Pseudomonas aeruginosa phage lyase (Endolysin) and its application as a bactericidal active component in medical hygiene, food processing and hygiene products. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa) is an important opportunistic pathogen that causes nosocomial infections, and is an important pathogen that causes death in patients with fibropneumonia, burn patients, AIDS patients and immunodeficiency patients. The bacterium widely exists in various environments, and its adaptability is very strong, which can resist the action of fungicides and other antibacterial drugs. In recent years, especially with the emergence of multidrug-resistant strains, the treatment of infections caused by the bacteria has become more difficult. Therefore, there is an urgent need to develop antibacterial drugs with a new mechanism of action to control an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/70C12N1/21A61K38/48A61K8/92A61K8/66A61Q19/10A61Q17/00A61P31/04A23L33/195
CPCC12N9/503A61K8/19A61K8/345A61K8/44A61K8/463A61K8/66A61K8/925A61K38/00A61Q17/005A61Q19/10
Inventor 杨洪江荆兆元何洋张粉姣
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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