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Method for screening SecA ATPase inhibitor

A reaction system and sample technology, applied in the field of biomedicine, can solve problems such as slow progress and no feasible solutions, and achieve low cost, high efficiency, and simple and reliable screening methods

Inactive Publication Date: 2011-10-12
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

However, the work is progressing slowly, and so far there are still no reports of feasible solutions

Method used

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  • Method for screening SecA ATPase inhibitor
  • Method for screening SecA ATPase inhibitor
  • Method for screening SecA ATPase inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Sample Screening

[0048] In this example, the SecA enzyme of Pseudomonas aeruginosa is taken as an example to illustrate the screening method of antibacterial drugs.

[0049] 1. Preparation of recombinant PaSecAN68 protein

[0050] 1. Construction of pET20b / pasecAN68 plasmid

[0051] Using Pseudomonas aeruginosa genomic DNA as a template, the complete sequence of pasecAN68 gene (SEQ ID No.3) was amplified by PCR method. The primer sequence is: upstream primer 5'GAGATATA TTTGCGCCTTTG 3'; downstream primer 5' ACAGCGCCTTCATGA 3'. The parts in italics are the enzyme cleavage sites of Nde I and HindIII, respectively.

[0052] The PCR reaction system includes:

[0053] Template (genomic DNA) 1 μl

[0054] Upstream primer (20pM) 1μl

[0055] Downstream primer (20pM) 1μl

[0056] 2×Pfu Mixture 25μl

[0057] nuclease-free ddH 2O 22 μl

[0058] Total volume 50μl

[0059] The conditions of the PCR reaction are:

[0060] Pre-denaturation at 95°C for 5 min...

Embodiment 2

[0104] Embodiment 2 antibacterial test

[0105] Strains:

[0106] Escherichia coli (Escherichia coli) BL21.19 / pET20b / PasecA: a temperature-sensitive Escherichia coli expression host cell BL21.19 that highly expresses the SecA protein of Pseudomonas aeruginosa, and the test bacteria have been preserved on October 16, 2006 In the General Microorganism Center of China Committee for Microbial Culture Collection (CGMCC, No. 1 Beichen West Road, Chaoyang District, Beijing, postal code 100101), the preservation number is CGMCC No.1838. Escherichia coli (Escherichia coli) BL21.19 / pET5a / EcsecA: a temperature-sensitive Escherichia coli expression host cell BL21.19 that highly expresses Escherichia coli SecA protein. General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, postal code 100101), the preservation number is CGMCC No.1839. BL21.19 / pET20b: temperature-sensitive Escherichia ...

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Abstract

The invention provides a method for screening a SecA ATPase inhibitor. In the method, an optimized ATPase activity measuring system is provided, and a protein level screening model of the SecA protein ATPase activity inhibitor is established. The method is simple and reliable, and has the advantages of high efficiency and low cost, thereby being an ideal antibiotic screening method. By utilizing the screening method, 3 active positive compounds which can inhibit SecA ATPase of pseudomonas aeruginosa are screened out from 3220 samples in a compound library, and 45 positive samples are obtained from 2096 crude extracts of microbial metabolism, wherein the screening positive rate is 0.9% (an inhibition ratio more than 30% is taken as the screening positive standard).

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a screening method for SecA ATPase inhibitors. Background technique [0002] Pseudomonas aeruginosa (PA), also known as Pseudomonas aeruginosa, is a representative species of Pseudomonas. Pseudomonas aeruginosa is a non-fermenting Gram-negative bacterium with low nutritional requirements that is widely distributed in various settings in the hospital. It can cause many types of infections, such as bacteremia, pneumonia, intra-abdominal infection, wound infection, burn infection, urinary tract infection, etc. Due to the low permeability of its outer membrane and the existence of active efflux systems (Active Efflux Systems) that efflux various substances, most of them are naturally resistant to various antibiotic strains, and it is not easy to be controlled after infection. Pseudomonas aeruginosa is particularly potent in patients with complications, use of inserted catheters, and post...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/32G01N21/31
Inventor 余利岩王宇赵莉莉李秋萍魏玉珍
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI