Screening, purification and characterisation of antimicrobial peptides from Hermetia illucens

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Antimicrobial peptides (AMPs) are short proteins with antimicrobial activity. In insects, AMPs are secreted from cells and tissues and contribute towards innate immunity. Black Soldier Fly Larvae (BSFL), like other insect larvae express a diverse spectrum of AMPs to protect them against the microbes they encounter. IAMPs are attractive candidates to be developed as alternative antimicrobials, and novel alternatives to antibiotic growth promoters.Research Plan: To induce the production of AMPs, BSFL will be immunised with different foodborne bacterial pathogens. Following immunization, the BSFL will be reared for 24h. Immunized hemolymph will be collected and centrifuged to remove hemocytes and cell debris. The antimicrobial activities of BSFL hemolymph will be tested against a range of bacterial pathogens using an inhibition zone assay. FPLC and HPLC will be used for partial purification of AMPs from the immunised hemolymph. The antibacterial activities of the partially purified AMPs4against the bacterial pathogens will be measured by examining MIC. The amino acid sequence of the purified AMP(s) will be determined by Edman degradation. The molecular mass of the AMP(s) will be analysed and sequence validated by MALDI-TOF MS. Data will be compared to AMP sequences registered in GenBank using BLAST searches. During the project we expect to gain access to the completed genome sequence of BSF and we will interrogate the sequence data to identify the AMP(s). DNA fragment(s) coding the identified AMP(s) will be amplified by PCR and cloned into a pET expression vector. The recombinant plasmid will be transformed into Escherichia coli or another suitable host. Alternatively, we also have a cell-free system available if the AMP(s) kill all of the bacteria. The AMP will be expressed and purified using standard protocols. AMPs will be further charcaterised in various in vitro, and possibly in vivo assays.