Construction and Preliminary Expression of Eukaryotic Expression Vector of Hybrid Antibacterial Peptide Me-HNP1

Select partial gene sequences of Melittin and Human Defensin(HNP-1)for artificial fusion,and their physical and chemical properties,functional structure were predicted by bioinformatics. According to the Pichia pastoris codon preference principle,encoded the hybrid peptide Me-HNP1 gene,and polymerase chain reaction(PCR)amplified of the target gene and directional cloning to the shuttle type expression vector on pPICZa A. The constructed secreted expression vector pPICZαA-Me-HNP1 was transformed into Pichia pastoris competent cell GS115 by electroporation and induced to express with methanol as the inducer. The obtained crude protein was isolated and purified. Tricine-SDS-PAGE protein electrophoresis obtained a single clear band at 5 kDa. The antimicrobial peptide Me-HNP1 was successfully expressed in Pichia pastoris. Bioinformatics analysis revealed that the hybrid antibacterial peptide Me-HNP1 had 8 positive charges in its molecule,theoretical pI:9.55,aliphatic index:80.44 and a half-life of more than 20 hours in yeast. It showed that it had the potential to become an excellent antibacterial peptide,which laid the experimental foundation for the subsequent study of antimicrobial peptides.