Volume 25, Issue 5 (8-2018)                   RJMS 2018, 25(5): 9-18 | Back to browse issues page

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Cloning and expression of AcAMP Aspergillus clavatus in E. coli and investigate its antibody titration in mice. RJMS 2018; 25 (5) :9-18
URL: http://rjms.iums.ac.ir/article-1-5203-en.html
Abstract:   (3051 Views)
Background: Antimicrobial peptides have been considered as the key to change in the current antibiotics. AcAMP, Antimicrobial peptide is one of them, which has been purified from the Aspergillus clavatus ES1 fungus, and its biochemical properties and characterization indicate that this peptide has unique properties. The aim of this study was to express the gene of AcAMP peptide in Escherichia coli (E. coli) and investigate its antibody titration in mice.
Methods: In this experimental study, the AcAMP peptide-producing gene with BamHI and SalI enzymes was amplified from pUC57 containing gen using PCR and cloned to pET28a expression vector (+) and transformed into E. coli strain BL21 (DE3) in the next step. The expression of the AcAMP recombinant protein was induced by IPTG and purified by the affinity Ni-NTA chromatography. The recombinant protein was confirmed using Western blotting. Mice were intraperitoneally immunized with purified protein, and IgG titration was measured by ELISA method.
Results: The recombinant pET28a (+) expression vector was confirmed by PCR and sequencing. The 9 kDa produced protein was confirmed by SDS-PAGE and Western blotting. Antibody production process, using indirect ELISA, indicates the increasing trend of antibodies titration, especially after the fourth injection.
Conclusion: Purified recombinant AcAMP peptide and produced antibody can be used in antimicrobial research such as antimicrobial activity characterization of recombinant peptide and peptide identification in various applications.
 
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Type of Study: Research | Subject: Immunology

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