Screening And Optimization Of Antimicrobial Peptides Efficient Expression Strain In Cynoglossus Semilaevis

Antimicrobial peptide, a small molecule peptides with broad-spectrumantimicrobial activity, has been discovered about1900species, exists in almost allspecies.This polypeptide has many advantages,for instance,small molecular weight,good thermal stability, low immunogenicity, a wide range of anti-bacterial,not easy toproduce drug resistance, small drug side effects, and so on.Antimicrobial peptidehas important potential applications in the prevention and treatment of diseases ofhumans and animals. Our laboratory had been constructed the prokaryotic expressionstrain of Cynoglossus semilaevis. Based on this,we screened100recombinant strainsand try to find thebest expression conditions of hepcidin mature peptidein E.colibycomparing SDS-PAGE of expression protein. We also reserch the expression oftherecombinant strain in different conditions, including the adding inductor time,theinduction temperature,the induction time and the concentration of inducter.The bestadding inducter time is OD600=0.6-0.7,The best induction temperature is30℃，thebest concentration of inducter is1mM,the best induction time is10h.The laboratory has cloned Cynoglossus semilaevishepcidin and NK-lysinmature peptide-coding genes, and two forms of eukaryotic expression vectors wereconstructed. Hepcidin and NK-lysin mature peptide-coding genes were connected tothe secretary carrier pINA1317and intracellular expression vector pINA1312, and thetwo forms of expression vectors are transferred toYarrowia lipolytica Po1h.Thetworecombinantexpression vectorwere proved to beconstructed successfully bysequencing results and PCR amplification of yeast genomic sequences. Theexogenous gene expression of plasmids pINA1317and pINA1312don’t need anysubstance-induced because of the promoter hp4d. Meanwhile, Yarrowia lipolytica hasmany advantages such as high safety, low production cost, the advantages of theexpression of the protein activity.The construction ofYarrowia lipolytica expressionsystem of Cynoglossus semilaevis Gunther hepcidin and NK-lysin mature peptide coding gene will provide a theoretical basis and technical support for theresearchapplications of eukaryotic expression.