Cloning, Expression And Antimicrobial Mechanism Of Antimicrobial Peptide Oncorhycin Ⅱ In P.Pastoris

Antimicrobial peptides (AMP), a group of small peptides, widely exist in organisms and can be produced quickly when organisms receive excitation of extraneous materials or microorganisms. AMP have broad antibacterial spectrum and do not intend to induce drug-resistance bacteria, so AMP may become a new generation of antimicrobial agents and replace the application of traditional antibiotics in medicine, agriculture and food etc. The object of this study is OncorhyncinⅡ, an antimicrobial peptide isolated from rainbow trout (Oncorhynchus mykiss) skin secretions. OncorhyncinⅡconsists of 69 amino acid residues and has a molecular mass of 7.2kDa, with an isoelectric point 12.96. It is thermostable and is active against both Gram-(+) and Gram-(-) bacteria at submicromolar concentrations with no lytic effect on trout erythrocytes. Genetic engineering was used to clone and express the antimicrobial peptide in P. pastoris, the expressed peptide was isolated and purified, and the ant-bacterial mechanism of OncorhyncinⅡwas invesgated.1 Construction of the P. pastoris expression vector and the engineering strainThe cDNA of OncorhyncinⅡwas obtained from rainbow trout skin by using RT-PCR and cloned to pGEM(?)-T Easy vector. After the the sequence was confirmed by sequencing analysis, the mature OncorhyncinⅡgene was prepared by PCR using OncorhyncinⅡ/pGEM(?)-T Easy plasmid as template. The target fragment and plasmid pGAPZaA were digested by EcoRⅠand KpnⅠ, ligated with T4 DNA ligase, and the recombinant plasmid pGAOnⅡwere obtained. Plasmid pGAOnⅡand pGAPZaA was digested by BlnⅠrespectively and transformed into P. pastoris GS115 using electroporation method, and then the positive transformants were selected on YPDS plates containing ZeocinTM and by PCR method. The supernatant of fermentation was analyzed by Tricine-SDS-PAGE and antimicrobial activity assay. The result showed that OncorhyncinⅡwas expressed and secreted extra-cellularly with antibacterial activity, and the inhibition on Gram-positive bacteria was stronger than that on Gram-negative bacteria and fungi. OncorhyncinⅡalso has a strong thermal stability, and can withstand freezing and thawing treatment repeatly.2 Purification and propertie studies on OncorhyncinⅡFirstly, SP Sepharose Fast Flow was used to isolated and purified OncorhyncinⅡfrom fermentation culture, and three elution peaks were analyzed by Tricine-SDS-PAGE, the result showed that OncorhyncinⅡwas in elution peak 2 and the molecular weight of contaminant proteins were above 1.5kDa. Secondly, Membrane separation system (Mw:lOkDa) was selected to separate hybrid-protein and OncorhyncinⅡ. Finally, OncorhyncinⅡwas desalted by bag filter (Mw:lkDa) and the OncorhyncinⅡof high purity was obtained and freeze-dried. OncorhyncinⅡconcentration and MIC was analyzed by Bradford method and Tube dilution method. The result showed that MIC of OncorhyncinⅡagainst S.aureus bacteria was 0.54μg/L.3 The preliminary study of antimicrobial mechanism of Oncorhyncinll against S.aureusFirstly, the dynamics of OncorhyncinⅡagainst S.aureus bacteria was studied. To further characterize the antimicrobial activity of OncorhyncinⅡ, antimicrobial curve and transmission curve on S.aureus bacteria was drawn. It was showed that the ability of OncorhyncinⅡto kill S.aureus bacteria was effective and time-dependent, it might be through the "barrel-stave pore"mechanism that OncorhyncinⅡquickly penetrated and damaged the cell membrane.The main results obtained from the study:1. OncorhyncinⅡgene was obtained, the OncorhyncinⅡP. pastoris expression vector pGAOnⅡwas successfully constructed and OncorhyncinⅡwas expressed in P. pastoris expression system, the recombinant OncorhyncinⅡhad strong thermal stability and antibacterial activity. 2. Separation and purification procedure of the recombinant OncorhyncinⅡwas successfully established. High purity, good activity and stability of the recombinant OncorhyncinⅡwas obtained through this method.3. Antimicrobial effects and antimicrobial mechanism of OncorhyncinⅡwas preliminarily studied.