Expression In Escherichia Coli,Activity Of Antimicrobial Peptide Dybowskin-2CDYa Identified From A Chinese Frog(Rana Dybowskii)

Antibacterial peptide(ABP),is expressed by immune defense systems of all kinds of organism to resist pathogen and malignant cells.Antibacterial peptide is a small molecule peptide generally containing amino acids from 10-50,with the features of broad-spectrum antibacterial activity,strong thermal stability,small molecular weight and immunogenicity and the bacteria difficult to produce resistant.Such peptides are attracted increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics.The amphibian's skin is moist,nudity,so the skin glands secrete a series of antibacterial peptide to defense pathogen.They evolve an excellent chemical defense system in the course of resisting the invasion of the microorganism----Antibacterial peptide during long term of evolution.ABP is a small molecule peptide with the features of broad-spectrum antibacterial activity,stability,low antigenicity,and has little influence on normal cells.In addition,the antibacterial peptide causes the permeability of membrane increasing by interference the structure of the prokaryotic cells',so that has the antibacterial and bactericidal effect without drug-resistance.They may be developed into antibacterial drug.Dybowskin-2CDYa(accession no.:EU827809)(SAVGRHGRRFGLRKHRKH)is a novel cationic peptide which rich in arginineand identified from Frogs of R.dybowskii in our lab.D-2CDYa is different from other ranid antibacterial peptide reported,and it not only has special sequence and also more positive charge,high isoelectric point and strong hydrophilicityl.A kind of cation ?-helical conformation is present in the structure predictive analysis of D-2CDYa,the positive charge and Hydrogen Bond of arginineand which means its high affinity with bacterial membrane and strong antibacterial activity.In order to study the character and function of D-2CDYa,the primers were designed based on the sequence of D-2CDYa gene and its gene was cloned using SOE-PCR.The PCR product which encoded Dybowskin-2CDYa peptide was ligated into pET-30a and the recombinant plasmid pET-30a-Dybowskin-2CDYa was constructed.The recombinant plasmid transformed into BL21(DE3)plsS and were selected the recombinant strain by PCR.Positive transformation expression was induced by ImM IPTG at 37?.The cell was selected by induced 1?5h then analysed by SDS-PAGE.It showed that the peptide expressed exclusively at 7KDa.Using His tag antibody in WesternBlot,the target protein was expressed successfully.Enlarge culture the recombinant cell,induce expression,and collect the cell by ultrasonication at 4?.Centrifuge in 12000r/min,15min and the supernate was purificated by His-Tag Purification kit,then the protein was analysed by SDS-PAGE.,the purity can reach 90%.antibacterial activity analysis and hemolytic activity analysis showed recombination Dybowskin-2CDYa inhibited the proliferation of Gram-negative bacteria and Gram-positive bacterial.Conclusion:Dybowskin-2CDYa gene can be expressed correctly by using prokaryote expression system.The target protein were expressed analysis by SDS-PAGE and his tag antibody.Antibacterial activity analysis showed recombination Dybowskin-2CDYa inhibited the proliferation of Gram-negative bacteria and Gram-positive bacterial,and according to the result we can infer that the antibacterial activity on the Gram-positive bacteria stronger than the Gram-negative bacteria.Hemolytic activity analysis showed recombination Dybowskin-2CDYa had no hemolytic activity.The study provided the stable source of material for further study on the mechanism of antibacterial and supression of tumor cell,also accumulated research experience for altering amino acid sequence of the natural antimicrobial peptides.