Study On The Function Of Skin Antimicrobial Peptides Of Chinese Forest Frog, Plateau Forest Frog And Japanese Frog

Under the background of increasing emergence of resistance microorganism strains,development of new antibacterial drugs is on the agenda.Antimicrobial peptides(AMPs),with small molecular weight,encoded by specific genes,synthesised by ribosome,widely exist in the innate immune system of many organisms and generally have antibacterial activity.Besides broad-spectrum and highly effective antibacterial function,AMPs have the activities of anti-fungus,anti-virus,anti-oxidation,antitumor,immuno-regulation and so on.Also,AMPs are easy to put into production and not easy to develop resistance.So they have broad development prospects.In the long-term natural selection,frogs become the natural resources of AMPs as they can secrete a large number of AMPs from the skin glands.This study aims at researching the biological activities of the skin AMPs secreted from Rana chensinensis,Rana kukunoris and Nanorana pleskei,so as to lay the foundation for the research and development of new antibacterial drugs.In prophase researches,different AMPs cDNA sequences were obtained from the skin cDNA library of Rana chensinensis,Rana kukunoris and Nanorana pleskei,and their corresponding amino acid sequences were deduced.Some representative AMPs were synthesized,incuding 13 species:Brevinin-2CE,Chensinin-1CEb,Chensinin-3CE,Palustrin-2CE and Temporin-1CEe(Rana chensinensis);Brevinin-1Ka,Brevinin-2Ka,Chensinin-3Kb,Kukunorin-1K,Palustrin-2ka and Palustrin-2kb(Rana kukunoris),Pleskein-2NPL and Pleskein-3NPL(Nanorana pleskei).In this study,the biological functions of these 13 kinds of AMPs were studied.The main results are as follows:1.The antibacterial results showed that the majority of AMPs have broad spectrum antibacterial activities.The minimal inhibitory concentrations(MICs)of each AMP to wild-type E.coli and drug-resistant E.coli were basically consistent,and similar results were obtained in the experiments of wild-type and drug-resistant S.aureus.But the efficacy of most traditional antibiotics against the drug-resistant bacteria were lower than that of the wild strains,which suggesting that the antibacterial mechanism of AMPs is different from traditional antibiotics.2.Some AMPs showed prominent antitumor effect,especially the IC50(half inhibitory concentration)of Brevinin-2CE,Brevinin-2Ka,Kukunorin-1K,Temporin-1CEe,Palustrin-2CE,Palustrin-2Ka and Palustrin-2Kb to hematological tumor cells,like HL-60,K562,Jurkat,Raji and U937,were much lower than their HC50(half hemolysis concentration).It suggests that these AMPs have a more specific inhibition of hematological tumor cells.3.Most AMPes showed the DPPH radical scavenging effect except Palustrin-2Kb.But only a handful of AMPs could scavenge hydroxyl free radicals and have reducing power.In addition,Chensinin-1CEb,Chensinin-3CE,Chensinin-3Kb,Pleskein-2NPL and Pleskein-3NPL could scavenge DPPH radical,though they showed no antimicrobial and antitumor activities.4.Immuno-regulatory activities experiment results showed that besides Chensinin-1CEb,Chensinin-3CE,Chensinin-3Kb,Pleskein-2NPL and Pleskein-3NPL,the other AMPs could activate mast cells,leading to mast cells degranulation and the release some cytokines like ?-hexosaminidase and histamine which can regulate the activity of immune cells and improve the defence function of the immune system.In this experiment,the biological functions of 13 kinds of AMPs were detected in order to obtain the AMPs with application prospects in different fields and provide molecular templates for new drug research and development.