[Cloning, expression and characterization of a new hybrid AMP gene of Hex-Mag]

To enhance the antibacterial ability of Magaininl-12, its N side was joined with an alkaline peptide named Hexapeptide( RRWQWR), which would make Magaininl-12 cling to the membrane of bacterial cells even tighter. According to the partiality codon of Pichia pastoris, a new hybrid antibacterial peptide Hex-Mag was designed based on the sequence of Hexapeptide and Magainin( 1-12). Synthesized through gene splicing by overlap extension, the hybrid gene was cloned into pPIC9 to construct the expression vector pPIC9-HM. After restriction enzyme analysis and purification, the pPIC9-HM was transformed into Pichia pastoris GS115. And the positive clones screened by the phenotype were induced by methanol. After optimized the requirements for the flask-shaking culture fermentation, the hybrid antibacterial peptide was expressed on high level. The new peptide, which has a weight of 2.3kDa, could remain its inhibition activity after treating for more than 3 hours in boiled water. Detected by agrose diffusion assay, Hex-Mag showed its broad-spectrum antibacterial abilities not only to Gram-negative bacteria but also to Gram-positive bacteria. The function of additive positive charges were testified by the antibacterial experiments, and the results showed the activity of Hex-Mag was stronger than that of Magainin1-12 obviously.