Resources Discovery And Functional Research Of Antimicrobial Peptides

Antibiotics is the greatest discovery in the medicine history, which saved thousands of humans life and changed humans health greatly. But a terrible things has happened because of long term unreasonable and abuse. In clinic, more and more drug resistance bacteria have discovered, which are unsensitive to most of the antibiotics, we called them "Super bacterias". The speed of antibiotics research and develop is less than the speed of the occurrence of bacteria resistance to drug, so the number of useful antibiotics become less and less.Antimicrobial peptides was discovered in 1980s the last century, which have more advantages than antibiotics. For example, high speed of killing bacteria, broad antimicrobial spectrum and so on. But the most important advantages of antimicrobial peptides is the hard to occur resistance to drug. So antimicrobial peptides have the potential to substitute for other antibiotics.In our work, in order to look for more antimicrobial peptides, quantities of three amphibian species(Bombina maxima、B. microdeladigitora and Rana nigrovittata)、plant (Picea sitchensis) and human origin antimicrobial peptides were studied. The reaearch including antimicrobial activities、hemolytic abilities and structure analysis. Parts of them exert strong antimicrobial activities and weak hemolytic activities, so they have tremendous developing potentiality because of their clinical application. Also, the prokaryotic expression of human P-defensin give us resource for application as their high production and purity and simple purification procedure.It is well-known that there is a large amount of antimicrobial peptides in amphibian skins but few antimicrobial peptides are found in amphibian brains. Twenty-two and four antimicrobial peptides were purified and characterized from the brain homogenate of Bombina maxima and B. microdeladigitora, respectively. One hundred fifty-eight cDNA clones encoding 79 antimicrobial peptides were isolated from brain cDNA libraries of B. maxima and B. microdeladigitora. These antimicrobial peptides belong to two peptide groups (maximin and maximin H). Twenty of them are identical to previously reported antimicrobial peptides (maximin 1-8,10,11, maximin H1,3-5,7,9,10,12,15,16) from B. maxima skin secretions. Fifty-nine of them are novel antimicrobial peptides. Some of these antimicrobial peptides showed strong antimicrobial activities against tested microorganism strains including Gram-positive and-negative bacteria and fungi. The current diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal brains. The extreme diversity may give rise to interest to prospect the actual functions of antimicrobial peptides in amphibian brains.Two novel antimicrobial peptides with similarity to brevinin-2 family are purified and characterized from the skin secretions of the frog, Rana nigrovittata. Different from brevinin-2, which is composed of 33 amino acid residues (aa), both brevinin-2-RN1 and-RN2 contain 30 aa. Five cDNA sequences (Genbank accession numbers, EU136465-9) encoding precursors of brevinin-2-RNl and-RN2 were screened from the skin cDNAlibrary of R. nigrovittata. These precursors are composed of 72 aa including a predicted signal peptide, an acidic spacer peptide, and a mature brevinin-2-RN. Both brevinin-2-RN1 and-RN2 showed strong antimicrobial activities against gram-positive and gram-negative bacteria and fungi. The current work identified and characterized two novel antimicrobial peptides with unique primary structure.Two antimicrobial peptides (piceain 1 and 2) derived from sequences encoded Picea sitchensis are identified. Their amino acid sequences are KSLRPRCWIKIKFR CKSLKF and RPRCWIKIKFRCKSLKF, respectively. One intra-molecular disulfide bridge is formed by these two half-cysteines in both piceain 1 and 2. Antimicrobial activities of synthesized piceains against several kinds of microorganisms were tested. They showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and fungus Candida albicans but little antimicrobial activity against Bacillus subtilis. The results of nematicidal test showed they exerted strong nematicidal activities against Caenorhabditis elegans, following exposure for 5 h at concentrations as low as 10 ug/mL. They had weak hemolytic abilities against human and rabbit red cells. At the concentration of 250 p.g/mL, they induced red cell hemolysis of less than 5%. Circular dichroism Spectra of the two antimicrobial peptides were investigated in several solutions. Their main secondary structure components are beta sheet and random. The current work provides a novel family of antimicrobial and nematicidal peptides with unique disulfided loop containing nine amino acid residues. Piceains were identified as dual acting nematicidal and antimicrobial peptides which may provide great potential alternative to currently used nematicides.Human (3-defensin-2 (hBD-2) is a 41 amino acid cationic antimicrobial peptide displays lethal effects against yeast fungi, as well as a broad spectrum of pathogenic microorganisms. Since it was difficult for hBD-2 to produce immunogenicity in human body due to originated from human and for microbes to acquire resistance against it due to its special antimicrobial mechanism, hBD-2 has a great potential for medical use. To explore a economic approach for high expression of soluble active recombinant hBD-2 in Escherichia coli BL21(DE3) cells, cloned the chemically synthesized DNA sequence encoding mature hBD-2 into the pET-32a+vector and transformed into Escherichia coli to allow expression of hBD-2 as a His-tagged thioredoxin fusion protein. The fusion product was expressed with high solubility and purified on His-bind Resin. Recombinant hBD-2 was released from the fusion protein by formic acid cleavage and separated from the carrier thioredoxin by Sephadex G-50 and C18 reverse-phase HPLC. The molecular mass of the peptide determined by mass spectrometry is identical to that of native. The functional activity of the expressed hBD-2 was confirmed by a liquid growth inhibition assay. The peptide exhibited strong antibacterial activity against the bacteria Staphylococcus aureus (minimal inhibitory concentration (MIC) 37.5μg/mL) and Candida albicans (MIC 150μg/mL).Through upon four experiments, we find more resources for our antimicrobial peptides database. When we face the crisis of bacteria resistance to drug, we may have more choices.