Design and cloning of recombinant antimicrobial peptide Melittin-IMekn۲

Backgrounds: Today, using of antimicrobial peptides is an important issue due to the antibioticresistance of pathogenic microbes. Different organisms such as venomous animals produce awide range of these peptides. Antimicrobial peptides disrupt the integrity of cell membranes andinteract with intracellular targets. Peptide ۲۶ amino acids called Melittin isolated from the venomof honey bee, Apis mellifera and peptide ۱۳ amino acid called IMekn۲ were isolated from thevenom of Iranian scorpion Mesobuthus eupeus are two antimicrobial peptides that have beenproven to have antibacterial properties. IMekn۲ is similar to BmKn۲ that is an antimicrobialpeptide isolated from Mesobuthus martensii Karsch scorpion and shows strong antibacterialactivities against gram-positive and gram-negative bacteria.Materials and Methods: The aim of this study was construction and cloning of a fusionfragment containing Melittin and IMekn۲. For this purpose, the design and property of fusionpeptide was investigated by in silico studies such as ConSSert, RaptorX, ExPASY and HeliQuestservers. IMekn۲ and Melittin were amplified with special primers for each fragment andenzymatically digested.Results: The fusion fragments were ligated together by the ligase and then were amplified byPCR and cloned into prokaryotic expression vector called pET۳۲b (+). The cloning of Melittin-IMekn۲ was determined by colony-PCR and sequencing. Bioinformatics studies showed itshelical structure and hydrophobicity. In addition, the antimicrobial activities of Melittin-IMekn۲fusion peptide was also increased compare with two separate peptides.Conclusion: These results suggest that Melittin-IMekn۲ fusion peptide may be an effectiveantimicrobial agent for the treatment of antibiotic-resistant infections.

کلیدواژه ها:

Antimicrobial peptides ، Melittin ، Bmkn۲ ، IMekn۲ ، PCR