Identification and Molecular Cloning of A Bioactive Peptide from the Defensive Skin Secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea

Abstract

Amphibians have been living on the Earth for more than 370 million years. In the long history of evolution, amphibians have generated a unique self-defence system on the surface of their bodies - their skin secretion. There are literally thousands of active molecules in the skin secretions of amphibians and the bioactive peptides are predominant among those bioactive substances. In this study, the skin secretion of the Purple-Sided Leaf Frog, Phyllomedusa baltea, was harvested. The isolation and identification of bioactive peptides from its skin secretion were performed through both approaches of genomic and proteomic analysis. The mRNA was isolated from the skin secretion and used to establish a cDNA library by mRNA transcription. The target cDNA of interest was amplified and sequenced using a typical procedure of molecular cloning. On the other hand, MS/MS fragmentation sequencing was used to identify the amino acid sequence and posttranslational modification of the encoded mature peptide. Once the sequence was confirmed, the peptide was chemically synthesised and biological activity screening was performed subsequently. In this thesis, one bioactive peptide, named QUB2492, was discovered. The full-length nucleotide sequence of its precursor-encoding cDNA was identified and structural characteristics of the mature peptide were confirmed. Antimicrobial assays, anticancer assays and a haemolysis assay, were performed to evaluate the biological activities of QUB2492. It demonstrated that QUB2492 did not inhibit the growth of the three tested microorganisms and was not capable of inducing haemolysis, at a concentration up to 512 µM. Although, QUB2492 was failed to inhibit the growth of the other two cell lines, human melanoma cell MB435S and human prostate cancer, PC-3, it exhibited potent anticancer activity against the proliferation of two cancer cell lines including human non-small cell lung cancer cell, H157, and human VIII neuronal glioblastoma cell, U251MG. Practically, it inhibited the proliferation effect of H157 cells at the concentration of 10 nM. Therefore, this peptide is considered to have specific targets on lung cancer cells.

Date of Award	08 Sept 2017
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Christopher Shaw (Supervisor), Tianbao Chen (Supervisor) & Lei Wang (Supervisor)