| Peptide (antibacterial peptide), also known as anti-microbial peptides or peptide antibiotics are widespread in nature of a class of cationic peptides in cell bacteria, plants, insects, fish, amphibians and mammals are distributed, with Broad-spectrum bactericidal effect, and does not produce tolerance against pathogen invasion as the first line of defense is an important body of non-specific immune components. Fish antimicrobial peptides in international studies in recent years began, the first such Israeli academics from the Leopard sole Oren (Paradachirus marmoratus) isolated from a body of 33 amino acids peptide named pardaxin. This peptide with helix - coil - helix structure model, with more than melittin antimicrobial activity and lower activity of human red blood cell hemolysis, and its role with other natural antimicrobial peptides such as bombesin, sterilization and other phase peptide Ratio. Electron microscopy showed that the inhibitory mechanism of dissolution of the bacterial cell wall. The researchers then were different from the Winter Flounder, lampreys, catfish and other species of fish in different fish antimicrobial peptides isolated. Because fish is beginning to emerge adaptive immune defense of animals, the immune defense mechanism should adapt is not perfect, the fish live in water environment, there are a large number of bacterial pathogens, and thus the natural immune defense mechanism is very important. Research on fish antimicrobial peptides not only help to understand the immune mechanisms of fish in the day, while helping us find a new practice in the production of antimicrobial drugs, improve disease resistance of fish to provide a new way of thinking.In our experiment, antibacterial peptides were extracted from healthy carp(Cyprinus carpio L.) tissues such as skin, gill, liver, kidney with Methanol and Acetone. The extracts inhibited strongly the resistion of E.coli and Vibrio Anguiarum which are Gram-negative bacteria, but had little function on staphylococcus aureus which is Gram-positive. The antibacterial ability was different owing to different tissues. At the same time, the antibacterial activity of the extraction decreased by boiling for 10 min, while it increased when the sample was incubated with trypsin (0.25%) at 37℃for 1 hour. All the results suggest that different tissues of carp were identified to have the antibacterial peptides.In this study, based on a variety of animals, hepcidin cDNA sequence alignment results, based on their conserved sequence primers, using RT-PCR, cloned the cDNA fragment of carp hepcidin, and then by 5 `-RACE and 3 `-RACE technology is its full length cDNA sequence; using genomic DNA as template, was amplified by RT-PCR was carp genomic DNA sequence of hepcidin, its intron - exon structure was analyzed. The results showed that carp hepcidin cDNA full-length 647 bp, 46 bp from the 5 'untranslated region (5'-UTR), 276 bp open reading frame (ORF) and 325 bp 3 ' untranslated region (3'-UTR), of which 91 amino acid ORF region encoding a precursor hepcidin, from 24 amino acid residues signal peptide of 42 amino acid residues of the former domain and 25 amino acid residues of the mature peptide. Carp hepcidin mature peptide of 25 amino acids, containing eight conserved cysteine residues, 4 chain by a short hairpin disulfide bond formation, there is a special corner of disulfide bonds, which may be peptides Directly related to antibacterial activity. Full-length hepcidin carp genomic DNA 1209 bp, consists of three exons and two introns, encoding part of exon 1 5'-UTR, signal peptide and part of the former domain, exon 2 encoded part of the former domain Exon 3 encoded part of the former domain, the mature peptide and the 3'-UTR.Detected by RT-PCR, mRNA of the gene in normal carp liver, spleen, head kidney, foregut, hindgut, oral epithelium, gills, skin, muscle, gonads and other tissues of blood, using Real-time PCR technology Vibrio anguillarum was detected after stimulation with carp soaked liver, head kidney, foregut and hindgut in the mRNA level changes. RT-PCR analysis showed that, in normal carp, the highest level of hepcidin mRNA in liver, followed by spleen, hindgut, head kidney, anterior intestine, oral epithelium, skin, gonads and blood cells, low levels in the gill, and Were not detected in the muscle to its mRNA. Real-time PCR analysis indicated that Vibrio anguillarum immersion carp after stimulation, the overall level of the organizations of hepcidin mRNA increased first and then decreased, but the changing patterns of different organizations are not the same. Liver, head kidney and hindgut hepcidin mRNA levels 6 h after stimulation to the maximum, the liver was about 3 times the normal level, head kidney and hindgut can reach 17 times the normal level; foregut hepcidin mRNA levels in stimulated Began to increase after 6 h to 72 h to reach the maximum, about 8 times the normal level.To espress hepcidin in pichia, a shuttle vector pPIC9K, which can express fusion protein both in pichia and E.coli, was chosen. After transformation in E.coli, the constructed plasmid was extracted and linearized with sacI, then transformed int pichia via electroporation. The tansformed clone was plated on BMG medium, and the property of GS115 enabled only the transformed one could grow on the selective medium. PCR amplification also verified the hepcidin integration into pichia genome. Hepcidin protein was secreded into cultured supernatant under methanol induction. Recombinant hepcidin exhibited antibacterial activity against various Gram-positive strains and Gram-negative strains tested, but more effective to marine Vibrio.
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