Screening, purification and characterisation of antimicrobial peptides from Hermetia illucens

抗菌肽（Antimicrobial Peptides，AMP）是一类具有抗菌活性的短肽。在昆虫中，AMP从细胞和组织中分泌并有助于先天免疫。像其他昆虫幼虫一样，黑士兵蝇幼虫（BSFL）表达多种AMP，以保护它们免受遇到的微生物的侵害。IAMPs是一种极具吸引力的候选药物，有望成为抗生素生长促进剂的替代品。研究计划：为了诱导抗菌肽的产生，将用不同的食源性细菌病原体免疫BSFL。免疫后，将BSFL饲养24小时。将采集免疫后的血淋巴并离心，以去除血细胞和细胞碎片。将使用抑菌圈试验检测BSFL血淋巴对一系列细菌病原体的抗菌活性。FPLC和HPLC将用于从免疫的血淋巴中部分纯化AMP。部分纯化的AMP 4对细菌病原体的抗菌活性将通过检查MIC来测量。将通过Edman降解测定纯化AMP的氨基酸序列。将分析AMP的分子量，并通过MALDI-TOF MS进行序列验证。将使用BLAST检索将数据与GenBank中注册的AMP序列进行比较。在项目期间，我们希望获得完整的BSF基因组序列，我们将询问序列数据以识别AMP。将通过PCR扩增编码所鉴定AMP的DNA片段，并克隆到pET表达载体中。将重组质粒转化到大肠杆菌或另一种合适的宿主中。或者，如果AMP杀死所有细菌，我们也可以使用无细胞系统。将使用标准方案表达和纯化AMP。AMP将在各种体外和可能的体内测定中进一步表征。

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.