Artificial combined antistaphylococcus engineering multipeptide and its preparation method

A Staphylococcus, engineering technology, applied in the field of artificially combined antibacterial engineering polypeptide and its preparation, can solve the problem of only function

Inactive Publication Date: 2004-07-21
CHENGDU PHOTON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As mentioned above, colistin is an ideal ion channel antibiotic, but the disadvantage is that it can only act on Escherichia coli

Method used

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  • Artificial combined antistaphylococcus engineering multipeptide and its preparation method
  • Artificial combined antistaphylococcus engineering multipeptide and its preparation method
  • Artificial combined antistaphylococcus engineering multipeptide and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0015]In this example, the Agr D signaling polypeptide of Staphylococcus aureus is linked to the carboxyl terminus of colistin Ia to prepare a plasmid for artificially combining anti-Staphylococcus aureus engineering polypeptide, and the mutated plasmid is transfected into In the engineered bacteria with plasmids, the bacteria were mass-proliferated (4L FB medium, 225rpm, 37°C; 6h), and the bacterial cells were centrifuged (4°C, 6000g, 20min). Take 4°C, 50mM boric acid buffer + 2mM EDTA + 2mM DTT 50-80ml suspension cells, ultrasonic disruption (4°C, 40W, 1-2min), high-speed centrifugation to break up the cells (4°C, 50000-70000g, 1.5h), take the supernatant and add streptomycin sulfate to precipitate DNA, 4 ℃, 50mM boric acid buffer + 2mM EDTA + 2mM DTT2L dialyzed overnight, the supernatant was loaded on a CM ion exchange column, and after elution at 4 ℃, 0.3M NaCl + 50mM boric acid buffer, the anti-Staphylococcus aureus engineering polypeptide was obtained. Molecular weight 6...

Embodiment 2

[0017] The wooden embodiment is that the Agr D signaling polypeptide of Staphylococcus aureus is connected to the carboxyl end of the colicin Ia water-based pore structure domain, and the plasmid is prepared, and then through the technical steps used in the example one, a smaller than the example one is obtained. Anti-Staphylococcus aureus engineered polypeptide with a molecular weight of 20,000. In order to confirm the antibacterial ability of the engineering polypeptide, 5 μl (10 8 CFUS / ml) into 10ml of LB medium with glucose, incubated at 225rpm, 37°C for three hours, then added the antibacterial engineering polypeptide (final concentration 0.5μg / ml), continued to incubate at 225rpm, 37°C, and sampled every hour Spectrophotometer ((A595nm) colorimetric test, with oxacillin (Oxacillin, final concentration 5 μ g / ml) and Staphylococcus aureus without adding any drug as contrast (see attached figure 2 ), the results showed that: after the staphylococcus aureus in the control ...

Embodiment 3

[0019] In this example, according to the above technical route, we conducted an in vivo bactericidal experiment on the prepared 20,000 molecular weight anti-S. aureus engineering polypeptide. Eighteen Kunming mice, half male and half female, weighing 25-30 g, were intraperitoneally injected with penicillin-resistant Staphylococcus aureus (ATCC 29213, American standard strain, the lethal dose was calibrated and injected by the Pharmacology Office of Sichuan Antibiotic Research Institute, National Drug Inspection Bureau) 1 One hour later, they were randomly divided into 3 groups, (1) intraperitoneal injection of 0.5ml 0.9% normal saline, once every 6 hours, a total of 4 times as the control group (n=6); (2) intraperitoneal injection of 2.5mg penicillin G sodium, every 6 hours Once an hour for a total of 4 times (n=6); (3) Inject 2 mg of 20,000 molecular weight antibacterial engineering polypeptide into the intraperitoneal cavity, once every 6 hours for a total of 4 times (n=6). ...

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Abstract

An artificially recombined engineering polypeptide resisting against staphylococei is composed of the staphylococcus' signal transfer polypeptide and the colicin able to form ion channel or its water pore canal structure domain. Its preparing process includes using point mutation to insert the staphylococcus' signal transfer polypeptide gene into the site chosen by colicin gene loaded in engineering plasmid and transfecting the mutative plasmid to engineering bacteria. Its advantages are strong antibacterial power to staphylococei, and not generating resistance easily by staphylococei.

Description

1. Technical field [0001] The invention relates to an artificially combined antibacterial engineering polypeptide and a preparation method thereof, by preparing an engineered polypeptide (Engineered peptide) composed of different E1 family colicins that can form ion channels and a functional domain of a staphylococcal signal transduction polypeptide for antibacterial purposes. 2. Background technology [0002] Bacterial infection is still a major threat to human life and health. Since the advent of sulfonamides and penicillins, antibiotics have been invented by humans to achieve antibacterial effects by inhibiting bacterial cell wall synthesis, inhibiting or interfering with bacterial nucleic acid and protein metabolism and synthesis. Purpose, and then these antibacterial pathways are likely to induce bacteria to mutate and produce antimicrobial resistance. Therefore, people have been devoting themselves to the development of new antibiotics. The direct formation of ion ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
Inventor 丘小庆
Owner CHENGDU PHOTON BIOTECH
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