Activity, CDNA Cloning And Expression Of Antimicrobial Peptides From Skin Of Odorrana Schmackeri HSH

Antimicrobial peptides (AMPs) usually are comprise of less than 100 amino acid residues, rich with hydrophobic and positive charged amino acid residues. Rich diversity in the types, structure and mechanism of antimicrobial peptides make it very hard for microbial organisms to resist to them. New antimicrobial peptides discovering, existing APMs transformating and production process improving would become the most important research subjects in the field of novel antibacterial drug devolopment. Amphibian skin secretions contain many families of antimicrobial peptides, most of which have been found in the skin of species of the genus Odorrana. Odorrana schmackeris is widely distributed in the south area of the Yellow River. This study used Odorrana schmackeri HSH as material to try to discover novel AMPs resources. Results were as the following.Firstly, skin mRNAs from Odorrana schmackeri were extracted and then used as template to amplify their cDNA by RT-PCR.21 cDNA sequences, encoding a total of 11 peptides belong 4 families, were obtained, seven of which were novel AMPs. The seven novel antimicrobial peptides were coded as Odorranain-1hSc, Odorranain-2hSc, Esculentin-hSc, Nigrosin-hSc, Brevinin-2hSc, Brevinin-3hSc, and Brevinin-1hSc.Secondly, recombined Odorranain-1hSc was collected by prokaryotic expression and purification. Antimicrobial activity test showed that Odorranain-1hSc has significant activity against Candida albicans with the minimum inhibitory concentration (MIC) 25 u g.ml-1. The molecular weight of Odorranain-1hSc is 1772 Da by mass spectrometric analysis, which is identical to the result calculated by the amino acids sequence of the peptide.Finally, Brevinin-1hSc was obtained by chemical synthesis. Antimicrobial activity assay showed that Brevinin-1hSc have broad-spectrum antimicrobial activity. The MIC to Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis were 1.56 μg.ml-1 and 12.5μg.ml-1, respectively. The peptide has weaker activity against Candida albicans with the MIC 25 μg.ml-1. The MIC of Brevinin-1hSc against Gram-negative bacteria Escherichia coli was 12.5 ug-ml-1. In the Sterilization dynamics test, when the concentration of Brevinin-1hSc improved to be 6.25μg.ml-1, the Staphylococcus aureus could be clear away in 30 min. Though electron microscopy, vesicle-like protrusions were found on the wall of Staphylococcus aureus, its cell nucleus structure become dispersion, and pleat-like structure was detected in the cell internal after incubate Staphylococcus aureus with Brevinin-1hSc.