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Novel effective cerecidin and application thereof

A technology of lanthiobactin and lanthionine, which is applied to medical preparations containing active ingredients, applications, bacteria, etc., can solve problems such as time-consuming workload, and achieve simple yield, good bacteriostatic effect, and high antibacterial effect. The effect of bacterial activity

Active Publication Date: 2014-01-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method is mainly to screen the strains with naturally produced antibacterial active substances and obtain new lantibiotics through fermentation. This method is time-consuming and labor-intensive, and has a certain degree of randomness.

Method used

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  • Novel effective cerecidin and application thereof
  • Novel effective cerecidin and application thereof
  • Novel effective cerecidin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, construction and mutation of expression vector

[0063] 1. Extraction of Bacillus cereus genomic DNA

[0064] (1) Collect 1.0-1.5ml of Bacillus cereus AS1.1846 (Bacillus cereus AS1.1846) in the stationary phase of overnight culture.

[0065] (2) 1×10 4 rpm, centrifuge for 30-60s, and discard the supernatant.

[0066] (3) Use 100 μl of TES buffer containing 30 mg / ml lysozyme to resuspend the bacterial pellet, and let stand at 37°C for 1.5h.

[0067] (4) Add 600μl lysis buffer, invert the centrifuge tube, mix gently, and place at room temperature for 10-15min (until clear).

[0068] (5) Add 10μl proteinase K (10mg / ml) and place at 37°C for 15min.

[0069] (6) Heat at 80°C for 5 minutes and cool to room temperature.

[0070] (7) Add 200 μl of sodium acetate (3M, pH 5.2), mix well, and place on ice for 10-15 minutes.

[0071] (8) 4°C, 2×10 4 Centrifuge at rpm for 10 minutes to precipitate the protein completely.

[0072] (9) Carefully pipette the super...

Embodiment 2

[0104] Example 2. Induced expression and in vitro processing of proteins

[0105] 1. Pick a single colony of E.coli BL21(DE3) / pET28a-cerAM(E-1K) and inoculate it into 10ml LB culture medium (containing 50μg / ml Kan), and incubate overnight at 37°C with shaking.

[0106] 2. Transfer the cultured bacteria solution overnight to 1 liter of fresh LB culture solution (containing 50 μg / ml Kan) at a ratio of 1:100 by volume, and culture at 37°C and 220 rpm for about 2-3 hours with shaking. When the concentration of bacterial solution reaches OD 600 When it is 0.6-0.8 (mid logarithmic growth phase), add IPTG to a final concentration of 0.25-1mM.

[0107] 3. Adjust the culture temperature to 18°C, shake at 160rpm for 16-20h, then centrifuge at 5000rpm at 4°C for 20min to collect the bacteria, and the precipitate of the bacteria can be stored at -80°C for later use.

[0108] 4. The cells were thawed and resuspended in 25ml start buffer.

[0109] 5. After ultrasonically disrupting the c...

Embodiment 3

[0136] The antibacterial activity assay of embodiment 3, cerecidin

[0137] The antibacterial activity of cerecidin was determined by well digging diffusion method.

[0138] 1. Preparation of indicator bacteria plate:

[0139] (1) Cultivate Micrococcus xanthus NCIB8166 on a plate containing S1 medium, and place it in the refrigerator for about 20 days, pick a ring of Micrococcus xanthus NCIB8166 in normal saline, suspend and mix well, take 0.5ml of the suspension and add it to the melted 20ml of S1 solid medium was mixed evenly, poured into a petri dish with a diameter of 90mm, and laid flat to obtain an indicator plate of Micrococcus xanthus NCIB8166.

[0140] (2) Cultivate Enterococcus faecalis V583 (VRE) on a plate containing GM17 medium, and place it in the refrigerator for about 20 days, pick a ring of Enterococcus faecalis V583 (VRE) in normal saline, suspend and mix, take 0.5 Add the 20ml GM17 solid medium melted into 1ml suspension and mix well, pour it into a petri ...

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PUM

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Abstract

The invention discloses novel effective cerecidin and an application thereof. The cerecidin disclosed by the invention is a modified polypeptide. A synthetic method of the cerecidin disclosed by the invention is simpler compared with the conventional method. Meanwhile, the synthesized cerecidin has relatively high antibacterial activity, the antibacterial effect of the cerecidin for resisting vancomycin enterococcus faecalis V583(VRE) is better than that of nisin, and under a relatively wide pH condition and a high temperature, the cerecidin is relatively stable.

Description

technical field [0001] The invention relates to a novel high-efficiency lantibiotic cerecidin and its application. Background technique [0002] Lantibiotics are bioactive antimicrobial peptides produced by Gram-positive bacteria such as lactic acid bacteria, synthesized by ribosomes and post-translationally modified. They are considered as potential alternatives to antibiotics with strong antimicrobial activity against specific Gram-positive pathogens, especially methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, or neopenicillin-resistant bacteria active. As the clearest lantibiotic currently studied, nisin has been widely used as a natural and safe food preservative in more than 80 countries in the world due to its excellent antibacterial activity, and has been used for more than 40 years No serious bacterial resistance reaction was caused, and the application effect was ideal. However, nisin itself has some shortcomings in stress ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/335C07K1/22C07K1/16C12N15/31C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/70A61K38/16A61P31/04
CPCC07K14/32C12N15/70
Inventor 钟瑾王剑
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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