| Antimicrobial peptides are a recently discovered type of antimicrobial activity peptides and exist in organism and biosphere widely. They are important host-defense molecules of innate immunity and play a major role in innate and adaptive immunity. Most polypeptides share common features such as small size, cationic charge, an amphipathic nature and wide antimicrobial spectrum. Various insects are known to produce humoral antibacterial proteins in response to injection of dead or live bacteria. Insects produce a variety of antibacterial peptides which act synergistically to kill invading microorganisms. Antibacterial peptides are also found in other animals. They possess the activities of anti-plasmodium, and also act as tumcides. The insect immune system is a representative of the innate immune system also conserved in vertebrates. In Lepidoptera, from which the first antmicrobial peptide cecropin was purified, many antimicrobial peptides and proteins have been characterized, such as moricin and lebocins from Bombyx mori, and a defensin from Heliothis virescens. The propagation of immune-responsive cells in vitro has provided the basis for substantial contributions to our understanding of many aspects of the mammalian immune response. In contrast, the potential for exploring the innate immune response of insects using cultured cells is only beginning to be developed. Immune-reactive mosquito cell lines express various defensive factors, including transferring, lysozyme, cecropin, defensin, and prophenoloxidase activities. Many experiments have proved that many insect cell lines in vitro(Drosophila melanogaster, Sacrophage pergrime, Anthonomus grandis, Aedes albopictus, Trichoplusia ni) were induced by treatment with lipopolysaccharide, lamimarin, flagellin and heat-killed Escherichai coli can produce antimicrobial peptides. So, we induced antibacterial peptides from cultured cells.In our experiment, six cultured insect cell lines: Trichoplusia ni egg cell line(5B1), Spodoptera litura egg cell line(SL), Spodoptera frugiperda egg cell line(SF-9), Aedes albopictus blood cell line(C7-10), Pieris rapae cell line(Pr-49), Plutella xylostella cell line (Px) were chosen by our investigation. And the cultured cells were induced by 10-3mg/ml lipopolysaccharide, tryptophan, heat-killed Saccharomyces cervisiae and heat-killed Fusarium graminearum Schwabe and so on. We found that the antibacterial peptides were secreted to the culture medium. These antibacterial peptides wererecovered by acid precipitation then. By use of agarose diffusion assay and growth inhibition assay, the antimicrobial activity spectrum of the antibacterial peptide was defined. And we also compared the different effection of different induced time and different induced concentration. Our results are as following:(1) The result of activity assay of induced supernant from all cells showed that LPS was an effective inductor.(2) The result of activity assay of induced supernant from all cells showed that the induced production from 5B1, C7-10, SF-9 and SL cells' secretion supernatant can have activities against microbe, but those from Px and Pr-49 cells have almost no activeties. And the inhibitory activities comparison showed that the productions from 5B1 cells have the strongest activities.(3) The relation between the concentration of inducement and the activity of antibacterial peptides was detected. 5B1, C7-10, SF-9 and SL cells induced activities were promoted at 10"5 mg/ml -10°mg/ml LPS, among them, 10"3mg/ml LPS had the best induced effect to 5B1, SF-9 and SL cells, but lO^mg/ml LPS had the best induced effect to C7-10. If adding with fresh medium, all of them could recover from growth and proliferation. When the concentration of LPS came to 10°mg/ml, the cells were broken and dead.(4) Dynamical assay of induced activities for 5B1, C7-10, SF-9 showed: anti-microbial activities were promoted at 4-8h. And 5B1, SF-9 reached a peak point at 16h and then dropped gradually. C7-10 reached a peak point at 36h and then dropped. All of them dropped rapidly after 48h.(5) Results of antimicrobial activity spectrum assay indicated that antibacterial peptides had broad inhibitory activity against fungi and bacteria especially Gram-negtive bacteria.(6) The result of Tricine SDS-PAGE showed: 5B1 cells induced supernatant had one enhanced bands, the molecular weight of it was 5.2KD;C7-10 cells had two enhanced bands, the molecular weight of them was about 7KD and 5KD.
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