The Expression,Purification And Antimicrobial Activity Of Antibacterial Peptide Dybowskin-YJ And Ranatuerin-2YJ From Rana Dybowskii

According to the antibacterial peptides Dybowskin-YJ and Ranatuerin-2YJ sequences obtained in our previous work in the skin of Rana dybowskii.Using eukaryotic expression system Pichia pastoris to induce,express and purify antibacterial peptides.And the experiment of antibacterial peptide synergistic effect was carried out to evaluate the antibacterial and synergistic effects of the recombinant antimicrobial peptides.After total gene synthesis of Dybowskin-YJ and Ranatuerin-2YJ,double enzyme cutting was used to obtain target genes.Target genes were then connected to pPic9K plasmid to acquire recombinant plasmids of pPic9K-Dybowskin-YJ and pPic9K-Ranatuerin-2YJ.The recombinant plasmids pPic9K-Dybowskin-YJ and pPic9K-Ranatuerin-2YJ were transf-ormed into Pichia pastoris GS115 competent cells.Positive and highly expressed recombinant strains were screened out by the plate and PCR.The recombinant strain was induced by methanol,and the supernatant was detected by Tricine-SDS-PAGE electrophoresis to analyze the expression of target proteins.Using Ni-Agarose to separate and purify antimicrobial peptides and Tricine-SDS-PAGE electrophoresis to observe its purification effect.MIC of recombinant Dybowskin-YJ to E.coli and Staphylococcus aureus were 50 ug/mL,MIC of recombinant Ranatuerin-2YJ to E.coli and Staphylococcus aureus were 50 ug/mL.The recombinant antimicrobial peptides Dybowskin-YJ and Ranatuerin-2YJ have synergistic effect on the resistance of E.coli and Staphylococcus aureus.This experiment induced the expression of antimicrobial peptides Dybowskin-YJ and Ranatuerin-2YJ by using Pichia Pastoris expression system.The recombinant antimicrobial peptides Dybowskin-YJ and Ranatuerin-2YJ have effect on the resistance of E.coli and Staphylococcus aureus.