Gene Cloning Prokaryotic Experssion And Functional Analysis Of Antimicrobial Peptide Ceratoxin From Ceratophrys Calcarata

Amphibians as the type of transition from aquatic to terrestrial in vertebrate evolution, its skin exposed in damp living environment. In order to adapt to the environment, the skin has a variety of active polypeptides:antioxidant peptide to prevent long sunshine, strong ultraviolet radiation damage; antimicrobial peptides, as the important part of the innate immune,constitute the first line of defense to protect the body from microbial infection.Therefore, the skin of amphibians is the the treasure house of drugs with huge development potential.A255-bp cDNA encoding an84-amino acid residue (aa) precursor protein containing8half-cysteines is cloned from the skin of the frog, Ceratophrys calcarata. By sequence comparison and signal peptide prediction, the precursor was predicted to release a63-aa mature peptide. The predicted peptide named ceratoxin shares significant sequence similarity with toxin family of waprins containing the whey acidic protein-type (WAP) four-disulfide core domain found in snake venoms. It has been proved the common evolution of waprin and kunitz-like toxin families in venomous snakes. Kunitz-like toxins have been identified from amphibians but no amphibian waprin has been reported.Escherichia coli Rosetta-gami (DE3) and plasmid pET-32a(+) were used to express the recombinant ceratoxin.It was expressed as a fusion with a His-tag and proteolysised by enterokinase at23℃for16h to release from the fusion. The expressed ceratoxin was purified by AKTA Mono S and C8reverse phase high-performance liquid chromatography (RP-HPLC, Hypersil BDS C8,25×0.46cm) column. SDS-PAGE and Mass spectrometry analysis were also performed to identify the recombinant ceratoxin.The antimicrobial and trypsin-inhibitory abilities of recombinant ceratoxin were tested. Recombinant ceratoxin showed strong antimicrobial activities against wide spectrum of microorganisms including Gram negative and Gram positive bacteria and fungi. Its MIC against S. aureus (ATCC25923), E. coli (ATCC25922), B. subtilis (ATCC6633), and C. albicans (ATCC20032) was1.5,>250,5.9, and5.9μg/ml, respectively. It had no serine protease-inhibitory activity. The functional activity of ceratoxin is similar to that of the snake venom waprins. The current results indicate the presence of snake venom-like waprin with antimicrobial activities in amphibians, which possibly take part in the innate immunity of amphibians. In addition, the discovery of ceratoxin may shed light on the possible evolution connection between amphibian and reptile waprins.The study shows that ceratoxin can also be effective in killing Staphylococcus aureus in vivo by constructing the model of mice conjunctivitis caused by Staphylococcus aureus.the half-life of ceratoxin is about6h,which is safe to the body.This study provides a scientific basis for ceratoxin in clinical application.