Studies On The Relation Between Structure And Function Of RTCI, An Antimicrobial Peptide From Rana Temporaria Chensinensis, David

The war, between human species and thousands of bacterial species exists along the evolution process of human being, becomes more severity since the clinical application of penicillin at 1941. During recent sixty years, the exploration of drug-resistant bacteria and the discovery of novel antimicrobial agents have always been arresting, which is quite important for both human health and harmony environment..At present, there is one common difficult point on all the research of novel antimicrobial agents, that is, the speed of the research on the structure-function relationship of the agents is far lower than the number increasing of the agents. For example: until now, on the case of antibiotic, more than 5,000 antibiotic structures have been reported, only about 1,000 among them have been studied in detail and only 100 molecules have been translated to clinical practice; on the case of antimicrobial peptide, around 600 different sequences have been found, less than 100 of them have been clarified partly, only 3 peptides entered into clinical trial, but no one has been used as medicine. Comparing both of above antimicrobial agents(antibiotics and antimicrobial peptides), antibiotics are the primary tool against the environment microorganisms. During fighting with environment microorganisms, people have found the fact that the resistance of microorganisms to antibiotics is not been induced by antibiotics but it is that drug-resistant bacteira are enriched by the selective pressure of antibiotics. So, it is necessary to control the abuse of antibiotics to release the clash between bacterial species and human species. In recent years, multiple events related to antibiotic resistance microorganisms have been reported frequently, however, no news about antimicrobial peptide resistance microorganisms has been reported.Different with antibiotics, which are the second metabolic product of unicellular organisms, antimicrobial peptides belong to the natural immune system of multi-cellular organisms, based on the knowledge of now, unicellular organisms without the gene which associated with the resistance to antimicrobial peptides, that is to say antimicrobial peptides have the ability to inhibit the growth and reproduction of unicellular organisms (most of the microorganisms), but the microorganisms could not enrich the antimicrobial peptide resistance strains. Therefore, under the time to control the abuse of antibiotics to release the clash between environmental microorganisms and human beings, it has vital significance in the case of society and economy to research and development for antimicrobial peptides, and then put them into use.Considering about the current status of research on antimicrobial peptides, that is, the research on the structure-function relationship of the peptides is slower than that on the discovery of novel sequences. In this thesis, we focus on the research about the structure-function relationship of RTCI under physiological condition, an antimicrobial peptide purified from skin of Rana temporaria chensinensis, David.Chapter 3:The antimicrobial peptide RTCI was purified from the skin of Rana temporaria chensinensis, David.. The extraction buffer including 0.2 M pH 4.0 NaAc-HAc has been adopted to extract the sample from the skin, after that Sephadex G-25 and C18 RP-HPLC have been used to separate the RTCI from the other components. The pure elution peak of RP-HPLC and the single band on SDS-PAGE gel announced the high purity of the RTCI peptide. With the process of purification, the yield of the peptide RTCI is 0.09% of the total protein in the skin. The pure degree and the amount of the peptide purified here are just enough to do the further study.Chapter 4: The physiological function of RTCI to 8 strains of bacteria (including 36 MDR strains) has been studied, comparing with he time-Killer and peptide concentration-dependent experiment fact. The MDR used here were clinically separated by dept. of laboratory, China-Japan Union Hospital, Jilin University. The function of RTCI during both experiments has been found as sterilization, inhibition, nourishment or growth stimulation, which is quite complicated comparing with the functions of antibiotics. Unfortunately, in this study no straight evidence supports the relationship between the function mode of the antimicrobial peptide RTCI and the antibiotic resistant character of the MDR. That indicates the molecular mechanism of the physiological function might be different.Chapter 5: UV-visb, fluorescence, circular cichroism, infrared and Raman spectroscopy have been adopted to clarify the physiological structure of RTCI. The amino acid residues constructed RTCI is Tyr, Asx and Glx, and the infrared spectrum explored the peptide is a kind of negative charge antimicrobial peptide. It is suspected that the specific fluorescence spectra and electronic absorbance spectra are related to the strong hydrogen bond of Tyr phenolic hydroxyl groups and the side chains of Asn. Based on such unusual structure, RTCI exhibited a single emission band (λmax448nm), The peculiar fluorescence will provide the possibility for further studies of the antibacterial mechanism on molecular level.Chapter 6: A high quality cDNA library from the skin of Rana temporaria chensinensis, David has been constructed. The titer of the cDNA library was confirmed to be about 2.4×105pfu ml-1, the recombination rate was 99.4 %. The constructed cDNA library can be used for further screening and cloning of RTCI and new antimicrobial gene in Rana temporaria chensinensis, David.