Expression And Bacteriostatic Activity Analysis Of Antibacterial Peptide Cecropin B Genes From Bombyx Mori And Drosophila Melanogaster

Cecropin B is a small protein with a molecular weight of about4kD, and it is awater-soluble and heat-stable peptide, which is easy formed in amphipathic α-helix.Cecropin B has many important functions, such as antibacterial and antifungal activities.Recently, it has also been found to have antitumoural activity and restrain HIV. Thesefunctions have made people more interest, because it is difficult to obtain purifiedantibacterial peptide from biotic tissues in large quantity, and the cost of chemicalsynthesis method is high.This study constructs prokaryotic expression vector and eukaryotic expression vector.First, tow kinds of cecropin B2between Bombyx mori and Drosophila melanogaster wereexpressed in E. coli. Also have expressed Bombyx mori Cecropin B2in insect baculovirussystem．The results obtained are as follows:1. According to E. coli gene’s translation of anticodon preference, we syntheticBombyx mori cecropin B (CecB2) and Drosophila melanogaster cecropin B (CecB2),through the method of overlapping PCR,and optimize them respectively.Tow kinds ofcecropin B2were recombined with pET32a to construct expression vector pET32a-BmCe-cBand pET32a-DmCecB. Then two vector were expressed in E. coli BL21(DE3) afterinduction with IPTG respectively. them product were measured by SDS-PAGE andWestern blotting. The expected specific band indicated that the expression of cecropin Bgene in E. coli BL21(DE3) was successful. After the separation and purification of thefusion protein, the His Tag was excised from product in its N termination by enterokinase.Integrated antibacterial peptide was obtained. Antimicobial activity analysis results showedthat the two products could restrain E.coli DH5α after treated with boiling water, and theantibacterial activity were significantly different between DmCecb2and BmCecb2. Whilethe concentration of the two proteins is both10mg/ml, the diameters of bacteriostasiscircles were15and8mm, respectively. The minimum inhibiting concentrations were0.05and0.1mg/ml, respectively. These results suggested that prokaryotic expression efficiencyand antibacterial activity of DmCecb2were higher than that of BmCecb2.2. Through Bac-to-Bac system, antibacterial peptide genes Bombyx mori cecropin B(CecB2) was expressed in insect baculovirus expression vector system, because of itsadvantages such as high-level expression, endotoxin-free, post-translational modifications and so on. BmCecB and BmCecB-EGFP were cloned into baculovirus transfer vectorpFastBacI respectively. Two recombinant plasmid pFastBacI-cecB and pFastBacI-cecB-E-GFP were transferred to E.coli competent cells DH10Bac. The recombinant bacmidsBacmid-cecB and Bacmid-cecB-EGFP were obtained. Both bacmid DNA were transfected intosilkworm cells mediated by lipofection. The recombinant baculovirus was obtained andtransfected into silkworm cells to express. In fluorescence microscopy, green fluorescenttransgenic positive cells were observed. Subsequently, the cells were broken by ultrasonic,the results of antimicobial activity showed that of BmCecB has significant antibacterialactivity. The results of our study can be used as the basis of the further research.