| Antibacterial peptide is a kind of resistance-infection-pathogen polypeptides, which is produced by the immune system and exist broadly in insects, plants, animals and human body in vivo. Antibacterial peptides could not only resist tumor cells but also bacteria, fungi, and virus in some way. Especially it could kill the multi-drug resistance bacteria and tumor cells without affecting human normal cells. Therefore, it is a novel kind of drug with tremendous developing potential, and a hot spot in the exploitation of antibacterial biological drugs presently. Musca and Larvae mainly live in the bad environment with corrupt matters, dejecta and garbage, which can spread many diseases owing to a lot of pathogen on their body surface. However, they could well survive with so many infection pathogens. This suggests that Musca have strong immunity to pathogens and could produce some high effective antibacterial matters. Muscae is a good resources of medicine because of its broad distribution, easy cultivation and low cost. There is no ready-made method for extracting, separating and purifying antibacterial peptides from Musca. So we need to establish a set of methods for screening unknown bioactive peptides gradually, which is a strong exploring and laboring research work.In this study, the cultivation method of Musca domestica and larvae was established, and the immunity was induced by injury infection to excrete more antibacterial active peptides. A broad spectrum and high effective natural antibacterial peptide was separated and purified by using high speed centrifugation grade sedimentation, high speed centrifugation ultrafiltration, Sephadex gel filtration and reverse phase high performance liquid chromatography (RP-HPLC) techniques. Its antibacterial activity, antibacterial spectrum and molecular structure were also studied to lay on some primary foundation for further exploiting its usage, establishing cDNA clone, and expressing Musca antibacterial peptides with genetic engineering technique.1. Establishing cultivation method for Musca domestica and larvae The stable cultivation method was established with repeat experiments because the cultivation of Musca domestica and larvae is the premise condition for extracting antibacterial peptides. The feeding conditions were 250C～280C room temperature, 60%~70% air humidity and some light time for normal growth of Musca domestica, while kept good ventilation, ample nutriment, 60%~70% air humidity and reasonable light for larvae growth.2. Separation and purification of Musca domestica antibacterial peptides Immune-induced Musca domestica and Larvae were grinded to powder with liquid nitrogen, then the tissues powder (1g) was mixed with 10ml buffer (0.1 mol/L pH6.8 Tris–HCl, containing 2.0 mmol/L EDTA, 0.86 mmol/L PMSF) to homogenate which centrifugated at 50 000g for 60min to remove high molecular protein and tissue residues, and different protein components were gradually separated by using Sephadex G50 and Sephadex G25 gel filtration sequencely, last purification antibacterial peptides was completed by RP-HPLC.Thirty one fractions of different components of antibacterial peptide were collected in step of Sephadex G50 filtration chromatography, and rough screening of antibacterial activity was investigated. The antibacterial activity was detected from fraction 15 to fraction 19 (retention time 75min~100min), among them the components of fraction 17 showed the highest antibacterial activity (retention time 85min~95min). To further analyze the antibacterial components of 27 fractions collected by using Sephadex G25 chromatography, the high antibacterial activity of components from fraction 15 and fraction 16 (retention time 95 min~105min) was found. The antibacterial samples that collected by Sephadex G50 were analyzed by cation exchange chromatography, and 11 fractions were collected. Fraction 5 and fraction 6 showed antibacterial activity. These results suggested that the antibacterial peptide was cation...
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