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Published on: March 22, 2012
Immunological detection of AcAMP antimicrobial peptide secreted by Aspergillus clavatus
Ehsan Zamani1, Jamil Zargan1, Hossein Honari1
1Department of Biological Sciences, Faculty of Sciences, Imam Hossein University, Tehran, Iran.
Background And Objectives:
Aspergillus clavatus antimicrobial peptide (AcAMP) is a fungi-derived peptide with a broad spectrum of activity against pathogenic bacteria and fungi. Natural antimicrobial peptides, including AcAMP, have attracted many attentions in the development of new natural antibiotics against pathogenic bacteria, especially multidrug resistant ones.
Materials And Methods:
In the present study, acamp gene was codon-optimized and chemically synthesized in pUC57 cloning vector, subcloned into pET28a (+) expression vector and transferred into competent Escherichia coli BL21 (DE3) cells. The expression of AcAMP was induced by addition of Isopropyl β- d-1-thiogalactopyranoside (IPTG) and the expressed peptide was purified by Ni-NTA. BALB/c mice were immunized with the purified peptide and the ability of the immunized mice sera for the detection of the native AcAMP secreted by A. clavatus IRAN 142C was examined through ELISA and Western blotting techniques.
Results:
Both ELISA and Western blotting demonstrated the ability of the sera of the immunized mice to detect the native AcAMP.
Conclusion:
The results of the present work show that the raised antibody against recombinant AcAMP can be used to detect AcAMP peptide, an issue which paves the way to develop detection kits for the detection of AcAMP-producing organisms, purification of this valuable peptide for further investigations.
Insights
Researchers developed a method to detect Aspergillus clavatus antimicrobial peptide (AcAMP) using antibodies raised against a recombinant version. This advancement aids in identifying AcAMP-producing organisms and purifying this valuable peptide for further research.
Area of Science:
- Microbiology
- Biotechnology
- Immunology
Background:
- * Aspergillus clavatus antimicrobial peptide (AcAMP) exhibits broad-spectrum activity against bacteria and fungi.
- * AcAMP is a promising candidate for developing novel antibiotics, particularly against multidrug-resistant pathogens.
Purpose of the Study:
- * To express and purify recombinant AcAMP.
- * To generate antibodies against AcAMP in mice.
- * To validate the use of these antibodies for detecting native AcAMP.
Main Methods:
- * Codon-optimization and synthesis of the *acamp* gene.
- * Expression of AcAMP in *Escherichia coli* and purification using Ni-NTA chromatography.
- * Immunization of BALB/c mice and detection of native AcAMP via ELISA and Western blotting.
Main Results:
- * Successful expression and purification of recombinant AcAMP.
- * ELISA and Western blotting confirmed that the generated mouse sera could detect native AcAMP secreted by *A. clavatus*.
Conclusions:
- * Antibodies against recombinant AcAMP are effective for detecting the native peptide.
- * This finding supports the development of detection kits for AcAMP-producing organisms.
- * Enables further purification and investigation of AcAMP's therapeutic potential.
