| Silkworm antimicrobial peptide from Hyatophora cecropia was discovered by H.boman for the first time in the 70s of the last century.It is known for low molecular weight, good thermal stability and water soluble.Because of their broad-spectrum antibiotic and the antibacterial mechanism which different from antibiotics, antimicrobial peptide are being increasingly recognized as potential candidate therapeutic drug against bacteria , tumor cell, and be applied in transgenic plant and animals. More deeply as the research to the structure, function and antibacterial mechanism of antimicrobial peptide, and more efficient and low noxious antimicrobial peptides were required by molecular modifications and designs. Thus, we could have a deep understanding to the relationship between the peptide structure and function.Most of the discovered antimicrobial peptides have plenty of positive charged amino acids, so they are cationic peptides constitutionally.The antimicrobial peptides are classified based on their structures of which there are four major classes:β-sheet,α-helical, loop, and extended peptides.No matterα-helical orβ-sheet, amphipathic is their common character. Amphipathicα-helical peptides possess many characters such as they are the most abundant, widespread in nature and broad-spectrum antibiotic; the peptide chains are short enough for chemical synthesis; they have therefore been the subject of numerous structure/activity relationship studies.So most of experiments to date have focused primarily on the amphipathicα-helical peptides.Although the mechanism of antimicrobial peptide are ambiguity, many experiments proved that the transmembrane channel/pores are formed by bundles of amphipathicα-helices, such that their hydrophobic surfaces interact with the lipid core of the membrane and their hydrophilic surfaces point inward producing an aqueous pore. This pore formationcan be confirmed by stepwise conductivity increases in channel measurement. The transmembrane pore formation involves the following steps: binding of peptide monomers to the membrane in a helical fashion followed by insertion of the helices into the hydrophobic core of the membrane. Progressive recruitment of additional monomers increases the pore size leading to leakage of cell contents and thereby death of the cells.According to positive charge quantity, hydrophobic property and peptide configuration, eight modified cecropin-XJ genes were designed: cecropin-M1, cecropin-M2, cecropin-M3, cecropin-M4,cecropin-M5, cecropin-M6, cecropin-M7, cecropin-M8. Among of them, cecropin-M1, -M2, -M3, -M4, and -M8 were amplified by PCR; and cecropin-M5, -M6, -M7 were synthesized by chemosynthesis. The modified cecropin genes were digested by endonuclease and cloned into plasmid pGEX-4T-1 to construct recombinant expression vectors. After that, the recombinant plasmids were transformed into E.coli BL21(DE3).The results of SDS-PAGE showed that all the fusion proteins were expressed effectively, only cecropin-M1 was expressed in inclusion body and the rest were all soluble proteins. The optimum induction time and IPTG concentration for the fusion protein were 4h and 5mM, respectively. The fusion protein were purified by GST affinity resin, after freeze drying, the purified fusion protein was obtained.The properties of cecropin-XJ fusion proteins were investigated by antimicrobial assay in vivo and MIC (minimum inhibitory concentrations).The results showed that all the fusion proteins could inhibit the growth of the E.coli BL21, and the maximum antimicrobial activity was provided with cecropin-M4 and cecropin-M8. The activity of the two reached up to 0.16mg/mL and 0.19mg/mL, respectively, which is three times higher than that of the cecropin-XJ gene. While the activities of cecropin-M6 and cecropin-M7 both were decreased dramatically. These findings could provide the theoretical basis to further investigate the antimicrobial peptide molecular design.
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