Artificial combined antibacterial engineering polypeptide and its preparation method

An engineering and artificial technology, applied in the field of artificially combined antibacterial engineering polypeptides and its preparation, can solve problems such as slow progress

Inactive Publication Date: 2002-03-27
四川新泰克控股有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research in this direction has been progressing slowly. For example, the signal transduction polypeptide of Staphylococcus aureus, Agr D, was finally found out to be an octapeptide until 1995 (Ji et al, Cell density control of staphylococcal virus mediated by an octapeptide pheromone. Proc Natl Acad Sci USA.92: 12055-12059 (1995)), found in 1999 that adding this synthetic octapeptide in the early or middle and late logarithmic phase of Staphylococcus aureus can stimulate or inhibit the growth of Staphylococcus aureus. Growth (Mayville et al, Structure-activity analysis of synthetic autoinducing thiolactone peptides from staphylococcus aureus responsible for virus. Proc Natl Acad Sci USA. 96: 1218-1223 (1999))
[0004] As mentioned above, colistin is an ideal ion channel antibiotic, but its disadvantage is that it can only act on Gram-negative bacilli such as Escherichia coli

Method used

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  • Artificial combined antibacterial engineering polypeptide and its preparation method
  • Artificial combined antibacterial engineering polypeptide and its preparation method

Examples

Experimental program
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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 engineering bacteria with plasmids, multiply the bacteria in large quantities (4L FAB, 225rpm, 37°C, 6h), centrifuge and precipitate the bacteria (4°C, 6000g, 20min), take 4°C, 50mM boric acid buffer + 2mM EDTA + 2mM DTT 50 -80ml suspended cells, sonicate (4°C, 40W, 1-2min), high-speed centrifuge to crush the cells (4°C, 50000-70000g, 1.5h), take the supernatant and add streptomycin sulfate to precipitate DNA, 4°C, 50mM boric acid buffer + 2mM EDTA + 2mMDTT 2L dialyzed overnight, the supernatant was loaded on a CM ion exchange column, eluted at 4°C with 0.3M NaCl + 50 mM boric acid buffer to obtain an anti-Staphylococcus aureus engineering polypeptide, the molecular weight 63,000. In order to confirm th...

Embodiment 2

[0017] In this embodiment, the Agr D signaling polypeptide of Staphylococcus aureus is connected to the carboxyl terminus of the colistin Ia water-based pore structure domain, and a plasmid is prepared, and then through the technical steps used in the example one, it is smaller than the example one. Anti-Staphylococcus aureus engineering polypeptide with a molecular weight of 13,000. In order to confirm the antibacterial ability of the engineering polypeptide, 5 μl (10 8 CFUS / ml) into 10ml of LB culture medium, incubated at 225rpm, 37°C for three hours, then added the antibacterial engineering polypeptide (final concentration 0.5μg / ml), continued to incubate at 225pm, 37°C, sampled every hour by spectrophotometry Meter (A595nm) colorimetric test, ampicillin (Ampicillin, final concentration 0.5μg / ml) and Staphylococcus aureus-LB medium without adding any drugs were used as controls (see attached figure 2 ), the results showed that: after the Staphylococcus aureus in the control ...

Embodiment 3

[0018] This embodiment is based on the above-mentioned technical route. We connect the Agr D signaling polypeptide of Staphylococcus aureus to the amino terminal of the colicin Ia aqueous pore structure domain to prepare a plasmid, and then go through the technical steps used in Example 1 to obtain A smaller anti-Staphylococcus aureus engineering polypeptide with a molecular weight of 13,000 was obtained than Example 1. Preliminary experiments show that the polypeptide has the ability to resist Staphylococcus aureus.

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Abstract

The present invention discloses an artifically-combined antimicrobial engineering polypeptide. Said polypeptide is formed from pathogen signal transducing polypeptide and colicine capable of forming ion channel or its water porous channel structural domain. Its molecular structure is connection of carboxyl terminal of colicine or its water porous channel structural domain and amino terminal of signal transducing polypeptide or connection of its amino terminal and carboxyl terminal of signal trasducing polypeptide. molecular weight of the polypeptide is 10000-63000, it can be respectively connected with signal transducing polypeptide of different pathogen, and can form the engineering polypeptide resisting said pathogen. Said invented polypeptide preparation method utilizes the point mulation technique to insert the pathogen signal transducing polypeptide gene into the site selected by colicine gene loaded in the engineering plasmid, then transports the mutation plasmid into engineering bacterium to make production. It does not make bacteria produce resistance to drug, and can change signal tranducing polypeptide to produce any antibiotic.

Description

1. Technical field [0001] The invention relates to an artificially combined antibacterial engineering polypeptide and a preparation method thereof. The antibacterial purpose is achieved by preparing the engineered polypeptide (Engineered peptide) composed of different bacterial toxins and functional domains of signal transduction polypeptides. 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. However, these antibacterial pathways can easily 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 channels on the bacterial cell membrane to cause ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/245C07K14/31C07K14/315
CPCC07K14/315C07K14/3156C07K14/245C07K14/31A61P31/04
Inventor 丘小庆
Owner 四川新泰克控股有限责任公司
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