Identification of extracted antimicrobial peptide from hemolymph of Tenebrio molitor

Insect antimicrobial peptides are belonged to the innate immune system, which are produced in their fat mass and secreted into the hemolymph. The aim of this study was to isolate and purify the antimicrobial peptide from hemolymph of this insect and to determine the range of activity of the peptide. To increase the activity of insect immunity for the inducible expression of antimicrobial peptides, the suspension of Escherichia coli (ATCC۲۵۹۲۲) of ۱۰۸ cfu/ml was boiled for ۲۰ minutes, and washed twice with PBS buffer, then ۱۰۵ cfu/ml was inoculated to ۳۰ larvae of T. molitor and to ۱۰ larvae as control with the same volume of PBS buffer by G۳۰ syringe. At the time of injection and during hemolymph extraction, the larvae were inactivated by cold stress and ether. Then, ۱۸ and ۳۲ hours after injection, hemolymph was extracted in cold tubes containing anticoagulant buffer, then centrifuged at ۱۰۰۰۰ rpm (۳ to ۴ times), ۴ ° C, ۳۰ minutes. After extraction of hemolymph from other cell components, the proteins pattern in hemolymph of infected insects was compared with control using ۱۲% SDS-PAGE. To study the antibacterial nature of hemolymph, ۵۰μl of control and infected insect’s hemolymph was loaded on a well with ۵ mm diameter made on nutrient agar which were previously inoculated with ۱۰۵ cfu/ml of E. coli and Staphylococcus aureus (ATCC۲۵۹۲۳), and the zones of inhibition was measured by agar diffusion method after ۱۴ hours. Differences in ۳۰ kDa band width were observed compared to controls after ۳۲ h. The diameter of the inhibition zone for both bacteria increased about ۱۴ mm compared to the control (PBS). The steps of purification and separation of each protein from each set of proteins are underway to make a comparison with the same from local insects in Semnan

کلیدواژه ها:

Antimicrobial peptides ، Tenebrio molitor ، Hemolymph ، Infection