Gene Cloning, Expression Structure And Functional Cathelicidin-DM From Duttaphrynus Melanostictus

Cathelicidins are a class of host defense peptides, consist of a N-terminal conserved signal peptide, a prosequence "cathelin" and mature peptide at the C-terminus, ’which play an important role in natural defense system in most vertebrates.Cathelicidins not only possess broad-spectrum antimicrobial activity, but also possess many other biological activities, such as immune cell chemotaxis, inducing mast cell degrannlation and histamine release, angiogenesis, promoting wound healing, lymphocyte activation and cell apoptosis of variation. In addition, cathelicidins play an important role in curing cancer and repairing intestinal mucosa. Cathelicidins were found in mammals, reptiles, fish, birds and other animals, but they are not found in the Duttaphrynus melanostictus.A novel cathelicidins, named as cathelicidin-DM, was cloned from a cDNA library of the skin of D. Melanostictus. The Cathelicidin-DM open reading frame is made up of 164 amino acids encoding by 495 nucleic acid, including a signal peptide (21 aa), conservative cathelin area and cation mature peptide contains 37 amino acids (37aa), which contains 9 alkaline amino acid residues, and the isoelectric point is 11.11, the molecular weight is 4161.268 kDa. Sequence alignment showed that cathelin domain structure were highly conservative, and four conecutive Cys residue were also highly conserved in cathelin domain structure. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to analyze the gene expression of cathelicidin-DM in D.Melanostictus. All the tested tissues, including small intestine, large intestine, liver, spleen, ovarium and skin, could express cathelicidin-DM genes. But the expression quantity of organizations existed differences.Circular Dichroism revealed that cathelicidin-DM had a typical amphipathic a-helical conformation in the solution of TFE/H2O (9:1), besides, it was mainly formed with β-sheet and random coil in sodium phosphate buffer and sodium phosphate buffer SDS. Cathelicidin-DM displayed potent antimicrobial activities in vitro against a broad spectrum of microorganisms including MDR and XDR clinical isolates. The bacteria killing curve experiment showed that cathelicidin-DM could rapidly exert its antimicrobial activity. It just need to less than 15 min to kill 98% S. haemolyticus. Cathelicidin-DM had slight effect on hemolysis and hemolysis rate (< 5%) in human erythrocyte. In addtion, cathelicidin-DM had no inhibition effect on the growth of tumor cells, and it did not produce antidrug antibodies(ADAs) in mice. The structure characteristics of cathelicidin-DM and its strong antimicrobial activity maybe make it a template for the development of topical antibiotics.