Expression In Pichia Pastoris Of Antimicrobial Peptides From The Skin Of Rana Dybowskii And Its Antimicrobial Activity

Antimicrobial peptides(AMPs)which are pioneer effectors in natural immunity defense line,synthesize rapidly through the immune response inducted by pathogen.The AMPs has broad-spectrum antimicrobial activity.The type and quantity of AMPs from the amphibian skin and the secretion are richest in all Organisms.We use the Pichia pastoris expression system to express several AMPs from the skin of Rana dybowskii which is one of main amphibian species in northeast of china,they could become the experimental referece for the production of a new speciall antimicrobial drug,and be used to research the mechanism of antimicrobial effect and synergetic effect.Methods:Using the six recombinant pMD18-T as the template,six AMPs genes were amplified with PCR and double digested with EcoRâ… and Notâ… .These genes were ligated into vector pPIC9K digested with EcoRâ… and Notâ… .Then they were transformed into host strain GS115,several His+cell line was screened and multicopy transformants were screened by various G418 concentrations.The transformants were cultivated in flasks,and firstly used the nature EKR sequence in AMPs Precursors to get mature peptides in culture medium.The supernatant were desalted by Sephadex G-25,and then lyophilized.We tested minimal inhibitory concentrations(MICs)of AMPs on G⁺(Escherichia coli),G^-(Staphyloccocus aureus) in vitro,and they are combinated with Each other for the test of synergetic effect.The results were as follows:(1)we got six recombinant expression plasmids pPIC9K-DB₁,pPIC9K-DB₂,pPIC9K-DB₃,pPIC9K-CS₁,pPIC9K-CS₂,pPIC9K-AM₃,and got three recombined P.pastoris cell line which can successfully express AMP-DB₂,AMP-CS₂,AMP-AM3,respectively are five,eleven one transformants.The optimum condition is inducted by 0.5%methanol for 36 hour.(2)The antimicrobial effect on G⁺S.aureus of all AMPs is better than G^-E.coli,and the average MIC of G⁺S.aureus is 25%of G'E.coli.The mixture of AMP-DB₂ and AMP-CS₂can suppress G⁺E.coli better,the MIC is reduced by 75% and 50%respectively.The synergetic effect of AMPs on S.aureus is inapparent.Conclutions:This study has constructed P.pastoris expression system of AMPs from the skin of Rana dybowskii.The recombined AMPs have antimicrobial activity,and some of them displayed synergetic effect on G^-E.coli.