Studies On The Expression Of Antimicrobial Peptides Attacin In MDEC-07114Cell And Its Biological Activities

Objective: The antibacterial peptide attacin of Musca domestica was used as target gene,the eukaryotic expression system containing baculovirus shuttle vector was established byembryonic cell line (MDEC-07114) of Musca domestica, and it′s expression product wereused for research on bioactivity,include of antimicrobial, thermal stability, et al.Methods:1.The antibacterial peptide attacin gene of Musca domestica was artificiallysynthesized, to clone it into plasmid PUC57.The recombinant plasmid verified byrestriction endonuclease digestion and then connected it to pFastBacTMHTA donor plasmid,to recombinant baculovirus Bacmid DNA.2.Recombinant baculovirus Bacmid DNAtransfected Musca domestica embryonic cell MDEC-07114, extraction crude protein ofsupernatants and cell lysate to detected target protein by SDS-PAGE, then target proteinwas purified by nickel column.3.The expression of purified protein weredetected,identificated by SDS-PAGE and Western-Blot.4.Experimental study on biologicalactivity of expression product of antibacterial peptide attacin, include of the inhibition ofgram negative bacteria (Escherichia coli) and gram positive bacteria (Staphylococcusaureus, Bacillus subtilis), the tolerance and biological activity under water bath of60-100℃for1-30min and the acid-base interaction of pH5.0-10.0.Result:1.The baculovirus expression vector (Bacmid-pFastBacTM-attacin) with Muscadomestica of antibacterial peptide attacin was successfully constructed.2.After therecombinant baculovirus Bacmid DNA transfected the embryonic cells of Muscadomestica, cells appeared larger diameter, stop growing, separation phenomenon,et.al.3.The molecular weight of the protein were detected by SDS-PAGE analysis, which wereextracted from supernatant and culture cell lysate protein, and the target protein were bothdetected at24KD.4. The crude protein was purified by nickel column, then through theSDS-PAGE and Western-Blot test confirmed containing the target protein.5.Eexpression products can inhibit Escherichia coli、Staphylococcus aureus、Bacillus subtilis, and alsohas normal bioactivity to inhibit Escherichia coli under the acid-base interaction ofpH5.0-10.0and water bath of60-100℃for1-30min.Conclusion: The Musca domestica embryonic cell MDEC-07114can be used in thebaculovirus expression system to express the antibacterial peptide attacin gene; Theexpression products of antibacterial peptide attacin has biological activity.