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Anti-Pseudomonas aeruginosa medicine screening model using Pseudomonas aeruginosa efflux pump outer membrane protein MEXAB-OPRM as target

A technology of Pseudomonas aeruginosa and efflux pump, which is applied in the direction of biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems such as not seen in the screening model, and achieve the effect of overcoming the problem of high drug resistance of Pseudomonas aeruginosa

Inactive Publication Date: 2006-12-13
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 screening model has not been reported in domestic and foreign literature

Method used

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  • Anti-Pseudomonas aeruginosa medicine screening model using Pseudomonas aeruginosa efflux pump outer membrane protein MEXAB-OPRM as target
  • Anti-Pseudomonas aeruginosa medicine screening model using Pseudomonas aeruginosa efflux pump outer membrane protein MEXAB-OPRM as target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Cloning of embodiment 1 outer membrane protein mexAB-oprM operon gene

[0042] 1) Total DNA was extracted from wild Pseudomonas aeruginosa PAO1 by CTAB method:

[0043] Cultivate 5 ml of PAO1 suspension overnight according to conventional methods, and centrifuge for 2 minutes. Add 567 μl of pH 8.0 TE buffer to the pellet, resuspend, add 30 μl of 10% SDS solution and 6 μl of 20 mg / ml proteinase K solution, and bathe in water at 37°C for 1 hour. Add 100 μl of 5 mol / l NaCl solution to the mixture, then add 80 μl of CTAB / NaCl solution, and incubate at 65° C. for 10 minutes. Extracted twice with phenol / chloroform / isoamyl alcohol. Precipitate genomic DNA with isopropanol at room temperature.

[0044] 2) PCR amplification of the target gene:

[0045] The genomic DNA of Pseudomonas aeruginosa PAO1 obtained as described in step 1) was treated with restriction endonuclease HindIII and used as a template for PCR. According to the sequence data in the Pseudomonas aeruginosa ge...

Embodiment 2

[0046] Construction of the recombinant Pseudomonas aeruginosa overexpressing MexAB-OprM protein in embodiment 2

[0047] 1) The operon gene mexAB-oprM obtained in Example 1 is connected to the expression vector pAK1900:

[0048] After the target gene obtained in the cloning vector pUC18 obtained as described in Example 1 was excised with the restriction endonuclease HindIII, it was dephosphorylated and connected to the expression vector pAK1900 which was also treated and dephosphorylated by HindIII , the recombinant plasmid pAK1900-mexAB-oprM was obtained.

[0049] 2) Conventional methods of electric shock transformation of the host Bacillus XY229 (the four efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM are missing, kindly provided by Dr. Komori.Y).

[0050] 3) Prepare competent cells:

[0051] Inoculate single colonies into 25ml LB liquid medium, and culture overnight at 37°C with vigorous shaking; inoculate 1ml of the overnight culture into 50ml LB liquid medi...

Embodiment 3

[0053] Example 3 Preliminary screening model for anti-Pseudomonas aeruginosa drug screening with Pseudomonas aeruginosa efflux pump outer membrane protein MexAB-OprM as the target

[0054] components

Growth control group (C)

Sample control group

(S')

Sample Screening Group

(S)

MH liquid medium

+

+

+

1 mM IPTG

+

+

+

Adjuncts just below their MIC

Auxiliary antibiotics

+

+

+

1% Overnight culture of Aspergillus

+

-

+

Fermentation broth to be tested

-

+

+

[0055] Use the following equation to calculate the growth inhibitory effect of each concentration of auxiliary antibiotics on Pseudomonas aeruginosa:

[0056] R = { 1 - ( S 24 - ...

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Abstract

The invention relates to an antibacillus pyocyaneus drug screening model which selects bacillus pyocyaneus external discharge pump adventitial protein MexAB-OprM as bull's-dot and to a method of screening antibacillus pyocyaneus drug using the said screening model. The invention also relates to a recombination bacillus pyocyaneus of excess expression MexAB-OprM protein which can be used in the said screening model and the screening method. The invention also relates to the antibacillus pyocyaneus drug obtained by the said screening model and method.

Description

field of invention [0001] The invention relates to a drug screening model for anti-Pseudomonas aeruginosa with the outer membrane protein MexAB-OprM of the efflux pump of Pseudomonas aeruginosa as a target, and a method for screening drugs against drug-resistant Pseudomonas aeruginosa by using the screening model. The present invention also relates to a recombinant Pseudomonas aeruginosa overexpressing the MexAB-OprM protein used in the above screening model and screening method. The present invention also relates to the anti-Pseudomonas aeruginosa drug obtained by using the above screening model and method. Background of the invention [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa), also known as Pseudomonas aeruginosa, is an important nosocomial infection conditional pathogenic bacteria, ranking among the top three pathogenic bacteria of opportunistic infections, often infecting cancer patients, burn patients, etc. In immunocompromised patients, it can cause dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/18
Inventor 肖春玲杨丽霞田睿姚天爵刘艺霜游雪甫
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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