Molecular Cloning And Characterization Of Antimicrobial Peptides From Hylarana Guentheri Skin Of Hainan

Antimicrobial peptides(AMPs)are a group of cationic peptides coded by genes and synthesized by ribosmes with multiple activities.AMPs can kill or inhibit not only bacterial,isolated multiple-drug resistance strains from clinical,but also fungus,parasites,virus and cancer cellwhich have attracted the interest of the researcher.In order to adapt to the surrounding stressors,the defensive molecular-antimicrobial peptides play an important role in the innate immunity.Today more than 1500 different AMPs have been purified from amphibians.Hylarana guentheri(Ranidae:Hylarana)are widly distributed in South of China.According to Chinese Medicinal Animals Cyclopaedia,H.guentheri have the effects of promoting blood circulation and tissue regeneration,promoting reunion of fractured bones and drainage of pus,relieving pain etc,so it can be used in treatment of traumatic injury,fracture,inflamed wound.In this present study,several tests were performed to determine the activities of Hylarana guentheri skin secretion with the plate assay and colorimetric methods.Antimicrobial,catalase,antioxidant,casein hydrolytic,and trypsin inhibitor activities were detected.Subsequently,the antimicrobial bioactive compound was purified with reverse phase high performance liquid chromatography(RP-HPLC),then two major molecular peak(3,080.6 Da and 3,124.6 Da)were observed on the map of the Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS).A set of primers were designed according to the reserved sequences of AMPs obtained from amphibians,to amplified the cDNA sequences coding AMPs of H.guentheri.11 cDNA encoding dermal AMPs were identified(GeneBank accession number:KU518306-KU518316).The parameters of those putative mature peptides were predicted by Protparam tool.The calculated molecular mass matched well with the observed mass molecular from MS(the gap between the calculated mass molecular and the observed mass molecular results from intramolecular disulfide bond).Four temporin family peptides(temporin-GHa-GHd)were synthesized by chemical synthesis.The antimicrobial activities were tested using Staphylococcus aureus(ATCC 25923),methicillin resistant S.aureus(MRSA,ATCC 43300),and Bacillus subtilis(ATCC 6633),Gram-negative bacteria Escherichia coli(ATCC 25922),E.coli(Isolated strain,abbreviated as IS),E.coli(D 31),Vibrio alginolyticus(IS),Psewudomconas aeruginosa(ATCC 15442),and fungus Candida albicans(ATCC 10231).All four AMPs showed broad-spectrum antimicrobial activity and MIC were determined to each strains.Temporin-GHa-GHd were the most sensitive to S.aureus(ATCC 25923),the MIC value respectively are 6.8?6.8?12.9?12.7 ?M,they also showed exhibiton to S.aureus(ATCC 43300)with the MIC value range from 12.7-51.8 ?M.Temporin-GHa-GHd were stable at 25-100 ?,except temporin-GHb which the antimicrobial activity decreased approximated 50%at 80-100 ?.Pre-incubation of temporin-GHa-GHd at the range of pH 2.0-10.0 or salt solution(NaCl,0.5-2 mol),the antimicrobial activities were not influenced by the extreme external surroundings.Morphological observation of pathogenic E.coli(D 31)?E.coli(ATCC 25922)and S.aureus(ATCC 25923)treated with temporin-GHa-GHd at 370C for 60 min with scanning electron microscopy(SEM)showed that the surface of microbial cells were considerably rough and shrived.The mechanism of temporin-GHa-GHd against bacteria should be investigated further.