Study On Purification And Bioactivity Of Skin-antimicrobial Peptides In Rana Chensinensis

Antimicrobial peptide which was widely distributed in vivo, also known as peptide antibiotic, was a class of small peptide. Amphibian skin which could secrete large amounts of substances with antibacterial activity which the main component was antimicrobial peptide was moist and bare. This study was designed to extracted and isolated and purified the new antimicrobial peptides from the active substances of Rana chensinensis skin secretions. We would make a study of its biological activity, so that we can lay the foundation for the study of amphibian antimicrobial peptides.Experimental frogs had been induced by intermittent electrical stimulation and the skin secretions had been collected with ultrapure water. And then the skin secretions had been isolated and purified through high performance liquid chromatography and the product had been detected the antibacterial activity by S.aureus ATCC25923 as activity test indicator bacteria. Finally, we got one antimicrobial peptide which had a strong inhibitory effect. Through MALDI-TOF MS identification, we determined the antimicrobial peptide’s purity was over 95%. Sequence analysis indicated that the amino acid sequence of this antimicrobial peptides was FLPFFAACAITKKC which had a disulfide bond between the eighth and the fourteenth amino acid. We named it AMP-1.The antimicrobial peptide AMP-1 had been synthesized through solid-phase peptide synthesis method and then been purified. After antibacterial activity detection and mass spectrometry, we got the desired antimicrobial peptide AMP-1. MIC results showed that AMP-1 was not only against S.aureus ATCC25923 and E.coli ATCC25922 but also inhibit multi-drug-resistant strains. The results of hemolysis activity showed that the hemolytic activity of AMP-1 with the highest concentration was only 1.108%. Antitumor activity test results showed that AMP-1 had a strong inhibitory effect to human breast cancer MCF-7 cells and the inhibition rate of AMP-1 with the highest concentration was 75.8%. Besides, AMP-1 also could inhibit the growth of MCF-7 at the lowest concentration. Anti-toxoplasma activity test results showed that the inhibition of AMP-1 to Toxoplasma gondii could enhanced with increasing concentration and extension of time. 72 hours later, the inhibition rate of AMP-1 with 50μg/m L to Toxoplasma gondii was 72.5%, while the lowest concentration of AMP-1 still could inhibit Toxoplasma gondii.