Isolation And Purification, Biological Activity Of Antimicrobial Peptides From Skin Of Rana Guentheri

Rana guentheri was involved in this experiment. The first step, their crude extraction and secretion from frog were isolated respectively through four different inducement and extractionion ways. The results of their antibacterial activity in vitro showed that the crude extraction and secretion had antibacterial activity to Staphylococcus aureus, Aerom onas hydrophila, Vibrio anguillarum, but didn’t have obvious antibacterial activity to Candida albicans. Their antibacterial activity from strong to weak as followe: the frog skin secretion after electric shocks > the frog skin secretion after injection by norepinephrine > the frog serum induced by inactivated bacteria > the frog lixiviate substance of skin, MIC magnitude of skin secretion and extraction and blood serum is from 32 to 128μg/mL.The second step, the frog skin secretion after electric shocks which was the strongest antibacterial activity was used. Through Sephadex G-50 gel filtration to do the preparatory purification, then, using C₁₈ preparative RP-HPLC columniation by linear gradient elution to do further isolation and purification. According to the results of the RP-HPLC chart, per-eluate was collected. Freeze-dried by machine. Then the antibacterial activity of each eluate freeze-dry powder was tested by paper strip method to choose the strongest antibacterial powder. Using the C₁₈ RP-HPLC analysis its purity, Coomassie brilliant blue G-250 analysis its component, Tricine-SDS-PAGE detected molecular weight. The results show that: protein content of the strongest antibacterial powder close to 100%, its molecular weight is about 3800 Da. Because it has strong antimicrobial, We named it as antimicrobial peptide GUE I.The third step of experimental, detected amino acid sequencer of antimicrobial peptide GUE I by machine. The results show that: the peptide composed by 34 amino acid, sequence is FWFLDILNKTLGKIVNLFAGHLIKCTKVLTCPLP, molecular weight is 3858.789 Da. Import the sequence to antimicrobial peptide database （http://aps.unmc.edu/AP/main.php） to analysis and compared the sequence, found that compared with antimicrobial peptides RANATUERIN 3 sequence, they sequence similarity is 57.89%, and compared with RANATUERIN 2 sequence, the similarity is 45.94%. Then, ANTHEPROT 5.0 software was used for two-dimensional spatial structure prediction. Detected antibacterial activity against seven kinds of common bacteria with double dilution, discover MIC of the peptide GUE I to gram-positive bacteria and gram-negative bacteria is from 8 to 16μg/mL, but antimicrobial activity to Candida albicans is still weak.Through above analysis, antimicrobial peptide GUE I may be an antimicrobial peptide of the Rana guentheri skin secretion, the result provided a certain foundation of the theory for the frogs antimicrobial peptide research and production.