Purification, Cloning And Antimicrobial Analysis Of Cathelicidin-AL From Amolops Loloensis

Cathelicidins comprise a family of antimicrobial peptides (AMPs) sharing a highly conserved cathelin domain, and play a central role in the innate defense against infection in most of vertebrates. In earlier reports, cathelicidins are only found in mammals, birds, reptiles and fish. This is the first time in the world that cathelicidin has been found in amphibians. They not only possess broad-spectrum antimicrobial activity, but also, they possess many other biological activities, such as immune cell chemotaxis, mast cell degranulation and histamine release, angiogenesis, wound repair, cytolytic activity, and so on.In this thesis, a series of experiments were carried out to study cathelicidin-AL of cathelicidins antimicrobial peptide family from Amolops loloensis.A novel cathelicidins, named as cathelicidin-AL, was purified from A. loloensis skin by one step of gel filtration and one step of RP-HPLC. Edman degradation method was used for the determination of its amino acid sequence. The determined sequence was RRSRRGRGGGRRGGSGGRGGRGGGGRSGAGSSIAGVGSRGGGGGRHYA, composed of48amino acid residues. By Matrix-assisted laser desorption/ionization time of fly mass spectrometry (MALDI-TOF-MS), the molecular mass of cathelicidin-AL was determined to be4453.286Da. A660bp cDNA sequence encoding the precursor of cathelicidin-AL was cloned from a cDNA library of A. loloensis skin. The deduced amino acid sequence of cathelicidin-AL precursor is composed of179amino acid residues, including a N-terminal signal peptide, a highly conserved cathelin prosequence and a C-terminal mature peptide. Reverse transcription polymerase chain reaction(RT-PCR) was carried out to analyze the gene expression of cathelicidin-AL in A. loloensis. Cathelicidin-AL expresses in most of the selected tissues, including stomach, muscle, heart, kidney, lung, intestine, spleen, liver, and it slightly expressed in skin and no expression in brain has been detected. Evolution analysis revealed that cathelicidin-AL share higher homology with mammals and reptilia than that from fish.The antimicrobial activities of cathelicidin-AL were studied. Cathelicidin-AL has a diverse range of antimicrobial activity. It is microbicidal against a lot of Gram-positive bacteria and Gram-negative bacteria. The results of transmission electron microscopy indicates that the cell shapes of cathelicidin-AL treated bacteria are significantly changed, the interface between the cell walls and membranes was blurred, and blebs are clearly visible on the outer membrane. Apparently, cathelicidin-AL severely destroyed the cell walls and membranes of bacteria.In addition, cathelicidin-AL exhibits no hemolytic activity and cytotoxicity which makes it a possible new antibacterial.