Abstract:  Insect antimicrobial peptides (AMPs) have wide antimicrobial spectrum. Cloning and prokaryotic expression of antimicrobial peptide TmAMP3 gene from Tenebrio molitor could provide a basis for further application of this antimicrobial peptide. TmAMP3 was amplified by RT-PCR using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank. The gene TmAMP3 was subcloned directionally into the prokaryotic expression vector pET-30a and transformed into Escherichia coli BL21. After IPTG introduction, fusion protein was detected in the supernatant of lysates by SDS-PAGE and purified with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices, and the fusion protein HIS-TmAMP3 was obtained. In the presence of IPTG, the growth of BL21 transformed by pET30a-TmAMP3 was inhibited. Under conditions of high temperature 100℃ for 10 h and strong acid or alkali, antibacterial activities of the fusion protein still existed. The minimum inhibitory concentration (MIC) of the fusion protein against five bacterial strains was also detected. The experiment offers a theoretical foundation to produce antimrobial peptides with stability on a large scale.

Key words:  Tenebrio molitor, antimicrobial peptide, high-level expression, protein purification, antibacterial activity