| Cathelicidin is a kind of antimicrobial peptide which is widely existed in vertebrate. T he precursor peptide of cathelicidin was composed of the signal peptide, a highly conserved domain cathelin and the mature functional peptide, as unique structural characteristics make them as a a separate type of antimicrobial peptides. Currently, cathelicidin has been found from mammals, fish, amphibians, birds, etc. and more and more cathelicidins are being discovered and studied. Many reports showed that cathelicidins have high efficiency and broad spectrum of antibacterial activity and immune regulating activity, and are an integral component of the body’s immune system. In addition, cathelicidins also pocess biofunctions such as inhibiting tissue damage and promoting wound healing and angiogenesis, binding endotoxin, etc. Cathelicidins have broad research value and application prospects.In this work, the c DNA sequence of a new kind of cathelicidin was obtained by gene cloning methods from Shaoxing duck. The sequence and phylogenetic, biological activity, secondary structure, mechanism of action of the peptide were investigated.Based on the cathelicidin c DNA sequences of reference jungle fowl, quail, ring-necked pheasants, pigeons, a series of specific primers were designed and synthesized, and 3’RACE-PCR method was carried out to obain the cathelicidin c DNA sequence of duck. A cathelicidin c DNA of 441 bp was cloned, and the deduced amino acid coding sequence is composed of 146 amino acids, which contains 17-amino-acid signal peptide, 98-amino-acids cathelin domain, and 20-amino-acid functional peptide. The mature functional peptide was named asdCATH and its sequence is KRFWQLVPLAIKIYRAWKRR. Sequence alignment and phylogenetic analysis showed that d CATH and other avian species have high homology in cathelicidin precursor peptide sequence, but are quitely different in the mature peptide sequence, indicating a certain kinship in the evolutionary process.Circular dichroism spectra showed that d CATH adopt an α-helix structure. The bacteria inhibition test showed that d CATH had high effective bactericidal activity against 8 kinds of representative test bacteria, and the average MIC value was 4 μM, and the MIC value of E.coli was even 2 μM. The damage effect of d CATH on mammalian cells was detected using human red blood cells and Ha Cat cells. The results showed that, the toxicity of d CATH was lowat low concentrations. With the increase of the concentration, the damage of the peptide to the mammalian cells was also greatly enhanced. However, the addition of 10% fetal bovine serum in the culture medium can significantly decrease the hemolytic activity of the peptide against human red blood cells and Ha Cat cells. The peptide dCATH has good salt and thermal stability, and the effect of cation and high temperature treatment on the activity of the peptide is very small. Compared to the salt and thermal stability, d CATH showed somewhat weaker enzyme stability. The peptide activity was significantly decreased after treated by four selected enzymes. In particular, MIC of d CATH was >128 μM after papain or proteinase K treatment. To study the inhibitory mechanism of d CATH, effect of d CATH on bacterial cell membrane was investigated by determination of the inner and outer membrane permeability, endometrial depolarization, scanning and transmission electron microscopy, and other tests. The results demonstrated that d CATH, like most of antimicrobial peptides, can change the permeability of the inner and outer membranes, which cause that intracellular material outflow and kill the bacteria. |
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