Cloning and Expression of a Musca domestica Defensin Gene in Pichia pastoris

A gene encoding antibacterial peptide defensin was obtained by reverse-transcription PCR(RT-PCR) from housefly(Musca domestica),and was cloned into the pMD18-T vector.After restriction enzyme analysis and DNA sequencing,the defensin gene was ligated with yeast expression vector pPICZα-A and transformed into the yeast strain GS115 by electroporation.The high-copy clones were selected by the YPDS plates containing different concentrations of Zeocin.The positive clones,of which the chromosomes were integrated with the defensin gene,were identified by the phenotype and PCR.They were induced by 0.5% methanol and the expression products were tested by Tricine-SDS-PAGE.The results of agar plate cavity diffusion assay and bacteria inhibition analysis indicated that the expression products exhibited the antibacterial activity against Staphylococcus aureus.