Expression Of Antibacterial Peptide Domesticin And MnALF4 In Pichia Pastoris And The Study Of Their Antibacterial Mechanism

The abuse of antibiotics has led to a surge in bacterial resistance,which is a serious threat to human health.Therefore,new antibiotic drugs are in urgent need to be developed.Antimicrobial peptides are short peptides that are the first line of defense against pathogens in the host's innate immune system.They have broad-spectrum bacteriostasis and are expected to be a substitute for traditional antibiotics.In this study,the Pichia pastoris eukaryotic expression system was used to express the Musca domestica antimicrobial peptide Domesticin and Macrobrachium nipponense antimicrobial peptide MnALF4,and antibacterial activities and antibacterial mechanism of the two antimicrobial peptides were studied.Firstly,the recombinant expression vectors pPIC9K-domesticin and pPIC9K-MnALF4 were constructed and transformed into Pichia pastoris KM71 competent cells by electroporation.After methanol induction,the expression products were detected by Tricine-SDS-PAGE and Western Blot,and the expressed products were purified by nickel column affinity chromatography.And then,the antibacterial and hemolytic activities of the purified recombinant proteins Domesticin and MnALF4 were detected respectively.The effects of Domesticin and MnALF4 on the morphology of Escherichia coli and Bacillus subtilis were observed by Cold Field Emission Scanning Electron Microscopy(SEM),which aims at to study their antibacterial mechanism.The results indicated that Domesticin and MnALF4 were successfully expressed in Pichia pastoris,and the molecular weight are 5.4 kD and 12 kD,respectively.Both Domesticin and MnALF4 showed significant inhibitory effect against 22 bacterial species including Dysentery bacillus(a pathogen).Scanning electron microscopy showed that Domesticin can break the bacterial cell membrane,causing the bacteria to shrink and dissolve and eventually to die;MnALF4 can destroy the bacterial cell wall,make the rod-shaped bacteria become spherical protoplasts,and then dissolve and break down to cause bacterial death,and it also can destroy the bacterial cell membrane,causing the solute to drain out and kill the bacteria.Hemolysis activity experiments showed that the hemolysis rate of Domesticin and MnALF4 on animacells was less than 2%.In conclusion,Domesticin mainly acted on the cell membrane and changed the membrane permeability to make the cell integrity lose,which eventually caused the bacterial death.MnALF4 takes the cell wall and cell membrane as common targets and plays the role of bacteriostatic.Domesticin and MnALF4 have the potential to be a new antimicrobial agent with a broad spectrum bacteriostasis and low hemolysis rate.