Recombinant Expression and Antimicrobial Assay of Hevein-like Peptide AbAMP1

Abstract:

The gene of hevein-like peptide AbAMP1 was cloned into the vector pPIC9 successfully. After linearized with restriction enzymes, the recombinant plasmid pPIC9-Ab was transformed into Pichia pastoris SMD1163 by electrotransformation. The transformants were screened on MD plate medium, and then identified using PCR method. The transformant AS16 with Mut＋ phenotype was used for inducing expression and other assays. A protein band of about 4.7 kD was detected on the gel by a tricine-SDS-PAGE analysis of induced culture supernatant, which indicates the protein content of the supernatant of culture after 72 h induction are almost the target peptide AbAMP1. A conspicuous inhibition band formed on a double-layered B. thuringiensis-containing plate where AbAMP1 gel band located after acid-native PAGE. This suggested the recombinant expressed AbAMP1 retains its natural antimicrobial activity. The supernatant of induced AS16 culture showed remarkable inhibitory effect on Fusarium oxysporum f. sp. cubense and Bacillus thuringiensis, B.