Isolation, Purification, Molecular Diversity Of Antimicrobal Peptides From Secretions Of Amolops Loloensis Skin

Amphibian is a biological treasure with great potentials as well as important natural resources with economic and cultural value. Many bioactive compounds have been found in amphibian skin secretions. Their naked and wet skins are endowed with an excellent chemical defense system against microorganism infection. Bioactive peptides are important components distributed in amphibian skin glands. Antimicrobial peptides are an important part of the amphibian innate immune system against microorganism infection.Amphibian skin glands are rich resources for antimicrobial peptides. Amolops loloensis is distributed in mountainous regions of southwest China. In this thesis, the isolation, purification, molecular diversity and biological activities of antimicrobial peptides from skin secretions of Yunnan frog, Amolops loloensis was analyzed.In this study, five novel antimicrobial peptides belonging to two families were isolated and purified from the skin secretions of Amolops loloensis by Sephadex G-50gel filtration chromatography and C18reverse phase high performance liquid chromatography; the first family including two members is esculentin-2-AL (esculentin-2-ALa and-ALb); the second family including three members is temporin-AL(temporin-ALd to-ALf). The antimicrobial peptide family of esculentin-2is firstly reported in the genus of Amolops. Then, they were subjected to amino acid sequence analysis by automated Edman degradation. The result showed that the family of esculentin-2-AL is composed of37amino acid residues (aa); the family of temporin-AL is composed of16,13and10aa, respectively. By MALDI-TOF-MS analysis, the observed molecular weights were3866.42Da,3868.57Da,1596.72Da,1719.25Da,1659.29Da, respectively that matched well with the calculated molecular weights (3866.13Da,3868.11Da,1596.98Da,1719.03Da,1659.00Da).By molecular cloning, Ten different cDNA clones encoding the precursors of esculentin-2-AL and temporin-AL were screened and sequenced from the skin cDNA library of A. loloensis (GenBank Accession Numbers:EU311547-EU311556). These ten precursors share conserved signal peptide sequences composed of22amino acid residues (aa). All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. Esculentin-2-ALa,-ALb and temporin-Ald to-ALk were synthesized by solid phase synthesis on a peptide synthesizer. Diversity biological activities assay showed that most of them have broad-spectrum antimicrobial activity, only temporin-ALk has no antimicrobial activity against B. pumilus and B. cereus. Esculentin-2-ALs and temporin-ALs showed strong antimicrobial activities against S. aureus and C. albicans. The result of hemolytic activity indicated that most of the antimicrobial peptides showed a little hemolytic activity. At the concentration of100μg-mL-1, temporin-ALk showed the strongest hemolytic ability (25%) against rabbit red blood cell, but temporin-Alf showed hemolytic ability (only7%) against rabbit red blood cell. By Mega and DAMBE(Data Analysis in Molecular Biology and Evolution), the rate of nucleotide transition (ns)/nucleotide transversion(nv) of precursor was calculated. The result suggested that more transversion caused mutation of their precursor sequence. The rate of synonymous (ds)/nonsynonymous(dn) nucleotide substitutions of precursor, signal and propiece peptide and mature peptides were calculated. The result indicated that they might origin from the same ancestral gene, but undergo diff-erent selecting pressures.