Cloning of cDNAs encoding skin antimicrobial peptide precursors from Chinese brown frogs, Rana chensinensis and determination of antimicrobial, anticancer and hemolysis activity / 生物工程学报

ABSTRACT

Amphibian skin antimicrobial peptides exhibit a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacterium and cytotoxic activity responsible for inhibiting the growth of cancer cells. In this present study, six cDNAs encoding antimicrobial peptide precursors were cloned from the skin of Chinese brown frog, Rana chensinensis by RT-PCR and 3'-RACE procedure and identified as preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, preprobrevinin-1CEa, preprobrevinin-1CEb, and preprochensinin-1, respectively. The nucleotide sequences of cDNA encoding 59-65 amino acid composed of 289-315 bp. Preprotemporin-1CEa, preprotemporin-1CEb and preprotemporin-1CEc are members of temporin family, which usually are short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and preprobrevinin-1CEb were identified as the members of the brevinin-1 family of antimicrobial peptides since both peptides contain "RANA box" that it's responsible for forming Cys-bridged cyclic heptapeptides at the C-terminal region of peptide. The nucleotide acid sequence and the deduced amino acid Sequence of preprochensinin-1 were not found to be identity with any known amphibian skin defensive peptides, so, preprochensinin-1 was identified as a novel peptide precursor. Four of bioactive peptides temporin-1CEa, temporin-1CEb, brevinin-1CEa and chensinin-1 were synthesized to investigate their antimicrobial, anticancer and haemolysis activities. The results showed that all of the synthesized antimicrobial peptides in this study inhibited the growth of the Gram-positive bacterium, and exhibited the anticancer activity against the growth of MCF-7 cells and HeLa cells. Analysis of the R. chensinensis bioactive peptides and their gene expression will be beneficial for preservation of this species.