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N-acyl homoserine lactonase and medicine thereof

A technology of acyl homoserine lactone and acyl homoserine, which is applied in the field of N-acyl homoserine lactonase and its medicine, and can solve the problems of life-threatening, difficult to cure, severe infection and other problems of patients

Active Publication Date: 2017-11-03
INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pseudomonas aeruginosa has a high degree of multi-drug resistance to antibiotics, often causing serious infections that are difficult to cure, posing a huge threat to the lives of patients

Method used

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  • N-acyl homoserine lactonase and medicine thereof
  • N-acyl homoserine lactonase and medicine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] This example is used to illustrate the purification process of AiiK protein.

[0031] Using the K. huagui DNA with the preservation number of ACCC 06121 as the amplification template, use the primer pair such as SEQ ID NO.3 and SEQ ID NO.4 to amplify to obtain the amplified product; use EcoRI and HindⅢ endonuclease The amplified product was digested and cloned into the expression vector pET32a to obtain the pET32a-aiiK prokaryotic expression plasmid; the above pET32a-aiiK prokaryotic expression plasmid was introduced into E. coli BL21 cells to obtain the E.coli BL21 / pET32a-aiiK prokaryotic expression strain .

[0032] The E.coli BL21 / pET32a-aiiK prokaryotic expression strain was shaken in LB medium (containing 100 μg / mL ampicillin) at 37°C and 180r / min until OD 600 ≈0.6, add IPTG with a final concentration of 0.5mM, and continue to cultivate at 25°C and 120r / min for 12h to obtain culture products. The control group was the culture product obtained by culturing the E. ...

Embodiment 2

[0036] This example measures the enzyme activity of AiiK at 25°C.

[0037] In this embodiment, N-decanoyl homoserine lactone (C 10 -HSL) was used as the substrate to measure the enzyme activity. According to the reaction system (N-HSL 2.5 μL, the final concentration of AiiK is 4 μg / mL, add 10 mM PBS to make the total volume 500 μL), add each component into a 2 mL centrifuge tube and mix well, react at 25 °C for 10 min, and replace AiiK with 10 mM PBS in the blank control group Protein, after the reaction, immediately add a volume of 55.5 μL of 20% SDS (final concentration is 2%) to the centrifuge tube to terminate the reaction; add ethyl acetate to the centrifuge tube and vortex extract the reaction product, the extracted reaction The product was concentrated and dried under flowing nitrogen, then redissolved in methanol, passed through a 0.22 μm filter membrane, and determined by HPLC. 10 -HSL content and calculate enzyme activity. The unit of enzyme activity (U) is to deg...

Embodiment 3

[0040] In this example, the effects of temperature and other enzymes on the activity of AiiK enzymes were determined.

[0041] According to the method described in Example 2, the enzyme activity of AiiK after incubation at 37°C and 45°C for 2h, 4h, 6h, 12h, 24h, and 36h (calculated as 100% of the enzyme activity at 0h) was measured respectively. For specific results, see Table 1.

[0042] According to the method described in Example 2, the effects of chymotrypsin, trypsin or proteinase K on the AiiK enzyme activity when the AiiK protein was treated for 30 min and 60 min respectively were determined. In the control group, 10 mM PBS was used instead of chymotrypsin and the enzyme activity was counted as 100%. The specific results are shown in Table 2.

[0043] Table 1

[0044]

[0045] Table 2

[0046]

[0047] In Table 1, when the AiiK protein was incubated at 37°C and 45°C for 2 hours, the enzyme activity remained above 86%, at 6 hours, the enzyme activ...

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Abstract

The invention relates to N-acyl homoserine lactonase and a medicine thereof. An amino acid sequence of the N-acyl homoserine lactonase and a sequence as shown in SEQ ID NO.1 are at least 90% homologous, and the amino acid sequence of the N-acyl homoserine lactonase has N-acyl homoserine lactonase activity. The invention further provides a nucleotide sequence encoding the lactonase, an expression vector and a heterogenous expression strain. The medicine can treat (pseudomonas aeruginosa) infection. The N-acyl homoserine lactonase catalyzes lactone bonds of N-acyl homoserine lactone to hydrolyze, so that signal molecules between pseudomonas aeruginosa are quenched, and quorum sensing is interfered and inhibited. Compared with other N-acyl homoserine lactonase, the N-acyl homoserine lactonase has wider substrate adaptability and more efficient degradation activity and better effects when serving as the medicine for treating pseudomonas aeruginosa infection.

Description

technical field [0001] The disclosure relates to the field of biotechnology, in particular to an N-acyl homoserine lactonase and its medicine. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a Gram-negative opportunistic pathogen that is widely distributed in nature and hospital environments, and is one of the important pathogens of nosocomial infections. Pseudomonas aeruginosa can cause infections in a series of organs such as wounds, eyes and lungs, especially people with low non-specific immunity are more susceptible to infection. Pseudomonas aeruginosa has a high degree of multi-drug resistance to antibiotics, often causing serious infections that are difficult to cure, posing a huge threat to the lives of patients. The production of pyocyanin, secretion of extracellular proteases, and formation of biofilm by Bacillus aeruginosa are regulated by the quorum sensing system, causing complex biofilm-associated Pseudomonas aeruginosa infectio...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21A61K38/46A61P31/04C12R1/19
CPCA61K38/465C12N9/18C12Y301/01081
Inventor 阮志勇董卫卫赵述淼赵君才王彦伟张琪江旭周义清孔德龙
Owner INST OF AGRI RESOURCES & REGIONAL PLANNING CHINESE ACADEMY OF AGRI SCI
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