Establishment and application of recombined aminoglycoside dual-function modified enzyme in vitro metabolic model

A technology of aminoglycosides and modified enzymes, which is applied in the field of in vitro metabolic models of modified enzymes to antibiotics, can solve problems that have not been seen, and achieve the effect of simple method and high degree of automation

Inactive Publication Date: 2007-11-28
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors of the present invention proposed for the first time to use the method of high performance liquid chromatography to evaluate the stability of the bifunctional modified enzyme of aminoglycoside antibiotics, and this method has not seen relevant reports at home and abroad

Method used

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  • Establishment and application of recombined aminoglycoside dual-function modified enzyme in vitro metabolic model
  • Establishment and application of recombined aminoglycoside dual-function modified enzyme in vitro metabolic model
  • Establishment and application of recombined aminoglycoside dual-function modified enzyme in vitro metabolic model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of embodiment 1 bifunctional modified enzyme gene aac (6')-aph (2 ")

[0041] (1) Extract total DNA from clinically isolated E.faecalis HH22:

[0042] Inoculate E.faecalis HH22 in Naoxintang medium containing 15 μg / mL gentamicin, shake and cultivate overnight at 200 rpm, centrifuge 2.5 mL of the bacterial liquid to enrich the bacterial cells, wash the precipitate twice with 500 μL STE, and then add 500 μL STE to refill To the suspension, add lysozyme to a final concentration of 2mg / mL, incubate at 37°C for 1h, then add 250μL of 2% SDS solution, shake and mix well, add proteinase K solution with a final concentration of 200μg / mL, incubate at 55°C for 30min, phenol / chloroform / isoamyl alcohol extraction until no denatured protein exists. Add 1 / 10 volume of 3M NaAc and an equal volume of isopropanol to the supernatant, and precipitate genomic DNA at room temperature.

[0043] (2) PCR amplification of the target gene:

[0044] The genomic DNA of E. faecalis HH22 ...

Embodiment 2

[0045] Cloning, sequencing analysis of the DNA fragment of embodiment 2 object

[0046] (1) Cloning the resulting bifunctional modified enzyme gene aac(6')-aph(2")TA into the pGEM-T vector

[0047] Purify the bifunctional modifying enzyme gene aac(6′)-aph(2″) obtained in Example 1 with a DNA purification kit, and use Taq enzyme to carry out an A-added reaction on the recovered PCR product, and then combine the A-added product with The pGEM-T vector was subjected to a ligation reaction to obtain a ligation product.

[0048] (2) Preparation of Competent Cells

[0049] Pick a single colony and inoculate in 2mL LB liquid medium, shake at 37°C and 200rpm overnight; inoculate 0.5mL overnight culture in 50mL LB liquid medium, shake at 37°C and 200rpm until OD 600 =0.3; the culture was ice-bathed for 10 min, centrifuged at 4°C and 4100 rpm for 10 min, and 0.6 times the volume of ice-precooled CaCl was used for precipitation 2 Wash once, then add 0.04 times the volume of ice-cold Ca...

Embodiment 3

[0052] Example 3 Construction of the bifunctional modified enzyme gene protein expression vector:

[0053] (1) The target fragment is connected into the expression vector

[0054] Using the enzyme cutting sites NdeI and XhoI introduced during the cloning of the target DNA fragment, cut it out from the pGEM-T vector, connect it to the protein expression vector pET-30a(+) after the same enzyme treatment, and transform it into the example The CaCl obtained by the method described in 2 2 The DH5α competent cells prepared by the method were coated with kanamycin-resistant plates, positive clones were selected, and the plasmids were extracted and identified by enzyme digestion, and sent to the sequencing company for sequencing.

[0055] (2) Transformation expression host

[0056] The Escherichia coli BL21 (DE3) competent cell prepared by the method described in Example 2 is transformed with the protein expression plasmid pET-30a-aac (6')-aph (2 ") that has been constructed; Coat k...

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Abstract

The invention provides a kind of reconstructive E.coli CGMCC1667 and reconstructive aminoglycoside enzyme of double functional modification induced by reconstructive E.coli CGMCC1667, and a kind of metabolism model of reconstructive aminoglycoside enzyme of double functional modification, which is disclosed as the application in selecting antibiotic of reconstructive aminoglycoside enzyme of double functional modification and inhibitor of reconstructive aminoglycoside enzyme of double functional modification. This model can research acylation and phosphorylation of antibiotic by reconstructive enzyme of double functional modification. It is charateristic in that it is convenient, quick, accurate and has high degree of automatization, and it can distinct original type and catabolite. It can not only selecting antibiotic of reconstructive aminoglycoside enzyme of double functional modification and inhibitor of reconstructive aminoglycoside enzyme of double functional modification, and also evaluate the stability of new medicine to enzyme of double functional modification.

Description

technical field [0001] The invention relates to an in vitro metabolism model of modified enzymes to antibiotics, in particular to an in vitro metabolism model of recombinant aminoglycoside bifunctional modification enzymes to aminoglycoside antibiotics and its application. Background technique [0002] Aminoglycoside antibiotics are a large class of pure natural or semi-synthetic polyvalent cationic compounds with clinical significance. With the clinical application of such antibiotics, bacterial resistance to them has gradually increased. The main mechanism is that bacteria produce modification enzymes to covalently modify antibiotics through O-phosphorylation, N-acetylation and O-adenylation. The antibacterial activity of such antibiotics is greatly reduced, or even invalidated. [0003] Aminoglycoside bifunctional modification enzyme AAC(6′)-APH(2″) is the most common modification enzyme in Gram-positive bacteria (mainly Enterococcus and Staphylococcus aureus), it has bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N9/10C12Q1/48
Inventor 游雪甫李聪然蒋建东杨信怡洪斌王跃明肖春玲孙承航
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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