Screening, purification and characterisation of antimicrobial peptides from Hermetia illucens

抗菌肽是一种具有抗菌活性的短蛋白。在昆虫中，amp由细胞和组织分泌，有助于先天免疫。黑兵蝇幼虫（Black Soldier Fly幼虫，BSFL）和其他昆虫幼虫一样，表达多种amp，以保护它们免受遇到的微生物的侵害。iamp是一种有吸引力的候选药物，可作为抗生素生长促进剂的新替代品。研究计划：为了诱导AMPs的产生，将用不同的食源性细菌病原体对BSFL进行免疫。免疫后，饲养24小时。收集免疫后的血淋巴，离心去除血细胞和细胞碎片。BSFL血淋巴的抗菌活性将使用抑制区测定法对一系列细菌病原体进行测试。FPLC和HPLC将用于免疫血淋巴中AMPs的部分纯化。部分纯化的amps4对细菌病原体的抑菌活性将通过检测MIC来测定。纯化后的AMP的氨基酸序列将通过Edman降解来确定。将分析AMP的分子质量，并通过MALDI-TOF ms验证序列。数据将与使用BLAST搜索在GenBank中注册的AMP序列进行比较。在项目期间，我们期望获得完整的BSF基因组序列，我们将查询序列数据以确定AMP。编码鉴定的AMP的DNA片段将通过PCR扩增并克隆到pET表达载体中。重组质粒将转化到大肠杆菌或其他合适的宿主中。另外，如果AMP杀死了所有的细菌，我们也有一个无细胞系统。AMP将使用标准协议进行表达和纯化。amp将在各种体外和可能的体内分析中进一步表征。

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.