| Paa robertingeri(Anura: Ranidae) is a member of Paa Dubois, which is distributed in Sichuan province, Yunnan province and Chongqin district city in southwestern of China. It is an endemic species in China, which is scarce and in critically danger. Cathelicidin is a novel family of antimicrobial peptides which plays great roles in the innate defense of vertebrates. Cathelicidins are characterized by a conserved N-terminal signal peptide, and a highly conserved cathelin domain followed by a highly divergent C-terminal mature peptide. The mature peptide is released by protease excision, they act as a first line of defense against invasion by pathogens and other risks. By far, they have been found from almost very kind vertebrates, including human, mammals, birds, reptiles and fish. However, there are only a few reports of cathelicidins in amphibians. In the past few years, just four species such as Amolops loloensis, Limnonectes fragilis, Paa yunnanensis, Rana catesbiana and Tylototriton verrucosus have been published on their study of cathelicidins. In this thesis,a serise of experiments were carried out to study the structure and function on the cathelicidins from Paa robertingeri.In this study, a c DNA library was constructed to clone the cathelicidins antimicrobial peptides’ gene from the skin of P. Robertingeri. By designing special primers and making use of semi-nested PCR, we reported the identification and characterization of two cathelicidins. The deduced mature peptides named cathelicidin-PR1 and cathelicidin-PR2 are composed of 29(RKCNLFCKAKQKLKSLSSVIG TVVHPPRG) and 25(KECKDYLCKLLMKLGSSSHIESIDP) residues, respectively. Phylogenetic analysis revealed that P. robertingeri cathelicidins had the highest homology with P. yunnanensis, they have the nearest genetic relationship fitting the species taxonomy. It formed a cluster with other frogs and clustered from mammalias, reptiles, birds and fishes, implying a slightly difficult evolution relationship between these species, which can serve as cathelicidin evolutionary proof.The molecular weight of cathelicidin-PR1(3195.88Da)and cathelicidin-PR2(2838.34Da) are small, and both of them have one disulfide bond. The CD spectrograms showed the structures of cathelicidin-PR1 and cathelicidin-PR2 in water solution are random coil,however showed α-helix in the SDS solution,the same as prediction. This particular structuce contributed to the bacterial killing process.The antimicrobial activity of chemically synthesized cathelicidin-PR1 and cathelicidin-PR2 were tested against 13 strains of bacteria and fungi, among which included some medical isolated drug-resistance microorganisms. The results indicated that the synthetic cathelicidin-PR1 can kill Gram-positive and Gram-negative bacterium, even some fungal species. The minimal inhibitory concentration(MIC) was 11.73 u Mol on six clincal bacteria strains, among which the MIC of some strains are less than ampicillin. Strangely, the cathelicidin-PR2 showed the very weak antimicrobial function. By antimicrobial and bacterial killing kinetic analysis, at the concentration of 5×MIC,cathelicidin-PR1 took less than 45 min to kill the Bacillus cereus clinical strain cells, even faster than ampicilli(1.5h). Furthermore,the cathelicidin-PR1 and cathelicidin-PR2 exhibited very low hemolytic activity against human erythrocytes, on the dose of 100μg/ml and 200μg/ml, cathelicidin-PR1 induced 3.87% and 1.78% human erythrocyte hemolyisisrespectively, cathelicidin-PR2 induced 1.12% and 2.01% human erythrocyte hemolyisis respectively. Both cathelicidin-PR1 and cathelicidin-PR2 showed little hemagglutinating activity with or without the presence of Ca2+. In addition, both of them exhibits low antioxidant activity, lysing 2.92% and 2.30% of DPPH radicals at 80μg/ml respectively. The results suggested that the cathelicidin-PR1 may serve as a template for the development of novel antibiotics. |
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