Gene Cloning, Prokaryotic Expression And Functional Characterization Of Immune Function Gene, Cathelicidin From Coturnix Coturnix (Phasianidae)

Cathelicidins are a family of structurally diverse antimicrobial peptides foundexclusively in mammals. They formed a family due to a highly conserved cathelin regionbetween signal peptide and C-terminal mature peptide. Currently, cathelicidins have beenfound in many species, and more and more cathelicidins are founding. Substantial evidenceindicates that AMPs not only possess direct antimicrobial activities but also have multipleimmunomodulatory activities. They play an important role in both the innate and adaptiveimmunity of the host. Besides, cathelicidin posseses additional biological functions includingwound repair mechanisms, chemoattractant properties, induction of angiogenesis, interactionwith LPS, and induction of cytolysis.In current work, the gene cloning and characterization of three avian cathelicidinorthologues, Cc-CATHs precursors from Coturnix coturnix were reported, and therelationship between quail cathelicidins and other known vertebrate cathelicidins wasanalyzed. Two of the three cathelicidin-derived antimicrobial peptides, Cc-CATH2and3were chemically synthesized and their antimicrobial activities were examined. The origin ofthe cathelicidin gene, one of the most important genes in host immune system, and themechanism of structure diversity and function differentiation were discussed.Three cathelicidins were identified from a constructed spleen cDNA library of C.coturnix, using a nested-PCR-based cloning strategy, designated Cc-CATH1, Cc-CATH2andCc-CATH3for clarity. The full-length of their cDNA sequences are545bp,530bp and555bp,respectively. The amino acid sequences of Cc-CATHs precursor are deduced, including a N-terminal signal peptide, a highly conserved cathelin prosequence and a C-terminal maturepeptide. Three mature antimicrobial peptides were predicted: Cc-CATH1(26amino acids),RVKRVLPLVIRTVIAGYNLYRAIKRK; Cc-CATH2(32amino acids), LVQRGRFGRFLKKVRRFIPKVIIAAQIGSRFG; and Cc-CATH3(29amino acids), RVRRFWPLVPVAINTVAAGINLYKAIRRK. Evolution analysis revealed that quail cathelicidins have high homologywith cathelicidins characterized in ring-necked pheasant (Phasianus colchicus) and chicken(Gallus gallus), they are considered to show evolutionary ‘closeness’. Besides, we can usequail cathelicidins as a label to estimate the genetic relationship of species. Chemically synthesized Cc-CATH2and Cc-CATH3exerted potent antimicrobialactivity, especially the clinically isolated (IS) drug-resistance strains. Cc-CATH2andCc-CATH3showed considerable reduction of cytotoxic activity compared to other aviancathelicidins. They also exerted a negligible hemolytic activity against human erythrocytes.Moreover, recombinant Cc-CATH2was produced in Escherichia coli. The purified peptidemaintained its broad and potent bactericidal activity. The discovery of quail cathelicidinsmake it an excellent template for the development of topical antibiotics to battle the continuedproblem of increasing anti-microbial resistance.