| Japanese eels (Anguilla japonica Temminck et Schlegel) is one of the most important economic freshwater cultivated species in China. Nowadays, the high density and intensive farming of eels often results in bacterial diseases outbreak. In order to save the economic loss, farmers sometimes have to use some antibiotics to cure the diseases. However, the use of antibiotics might result in producing drug-resistant bacterial strains, or restricting the growth of fish, or antibiotics residue in fish body. Recently, it has been reported that there are several important nonspecific immune effectors form fish, which has broad-spectrum and strong antibacterial ability and has been drawn more and more attention. The antibacterial peptides are one kind of important nonspecific immune factor; its function mechanism is different from antibiotics. Antibacterial peptides permeate the bacteria cell membrane led the inclusion of bacteria cell to leak out resulting in the dead of the bacteria. So, there is no question of the peptide-resistance. To this point, antibacterial peptide may be a potential source to replace traditional antibiotic to cure fish diseases. In this study, we compared the antibacterial activities of different tissues and organs of A. japonica and tried to purify a peptide from the liver.1. Comparison and analysis of the antibacterial activities in different tissues and organs, including liver, gill, spleen, bile, kidney and serum, of A. japonica. In this study, we obtained acid extractions of the tissues and organs from Japanese eels. Using agar diffusion method, we detected the antibacterial activity of those extractions against four common pathogenic bacteria (Enterobacter spp. B01, Edwardsiella tarda B09, Aeromonas spp. B18, and A. hydrophila B27) from the cultivated eels. The results showed that antibacterial activities of liver, gill, spleen, bile were obvious. The extractions of kidney exhibit antibacterial activity against B18, B27, and B01. The extractions of serum exhibit antibacterial activity against B18. The antibacterial activities of extractions from different tissues and organs are different to the same bacterium. The antibacterial activity of liver is the highest to all experimented bacteria. The antibacterial activity of extractions from the same tissues or organs varied to different bacteria. The acid extractions were further purified by the method of reverse-phase liquid chromatography on Source 5R RPC-ST-4.6/150 column (Amersham Bioscience, Sweden). The results showed that the killing indexes of the extractions from liver, kidney, gill, and bile were higher than 50% when eluted by the concentration of aceonitrile ranging from 19.4% to 35.4%, while the killing index of extractions from serum and kidney are lower than 50% when eluted by the concentration of aceonitrile ranging from 2% to 80%.2. The extractions of liver from Japanese eels were ultrafiltrated by cross flow filter of a 10 kDa fibre column. Antibacterial activity against four common pathogenic bacteria from eels, including B01, B09, B18, and B27, was detected by using agar diffusion method. The antibacterial activity of the extractions with molecular weight less than 10 kDa is higher than that of the extractions with molecular weight more than 10 kDa. The antibacterial activity of the extractions to B18 was the highest than other three bacteria. The extractions of liver with molecular weight less than 10 kDa were further isolated by using ionic-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase chromatography on Source 5R RPC. The isolated extractions with a concentration of 0.1 mg/mL exhibited a killing index of 99.4% to the gram-positive bacterium A. hydrophila B27. The molecular weight of the isolated extractions was about 2.5 KDa from the results of Tricine-SDS-PAGE analysis, but it was not a single protein when determined by matrix-assisted laster desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and needed to be further purified and analyzed.
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