Construction, Expression And Detection Of Antimicrobial Peptide Brevinin-2CE

The antimicrobial peptide brevinin-2CE isolated from Chinese forest frog skin, whichencoding using E. coli preference codons, was expressed as series of2-fold expression mannerthrough add met in the N, C terminus in E.coli BL21. The gene Fragment was prepared by genesynthesis method. The DNA sequence length was258bp after add restriction sites, and thensplitted into10overlapping16bp,45bp average length of single-stranded oligonucleotides usingGenedesign β3.0. The single-stranded oligonucleotides were synthesised by phosphoramiditetriester method using ABI3900DNA automatic synthesizer, were purificated by the syntheticproduct PAGE.The brevinin-2CE gene was synthesised by the two-step method and one-step based on PCRreactions, connected to the pET31b plasmid by T4ligase after digested by AlwN I and Xho I,transformed into E.coli BL21competent Cells prepared through CaCl2method by heat shocktransformation. The recombinant bacteria selected by ampicillin and verified through PCR methodvalidation. The positive clones were inoculated in LB medium, induced by IPTG when the brothOD600more than0.7. Four hours after induced by IPTG, the OD600significantly lower than the uninduced bacteria showwed that brevinin-2CE successfully expressed and has antibacterial activity.Two one-step,two two-step synthetic gene positive clones were sequenced, results showwed thatthe four clones sequenced, only W01correct sequence. W03contains one base error, W04contains one base error, W05contains two bases errors. Four sequenced clones contained1024ntoccered four errors, the error rate was3.91‰.The culture was added IPTG （100mM） to a final concentration of1mM, induced for37℃and4h, the SDS-PAGE result shown that KSI-2CE bands had appearred specific, the initial judgehad expressed fusion proteins. The E.coli cells were collected by centrifugation,and then lysed bysonication. Inclusion bodies were separated from bacterial cells and soluble protein bycentrifugation, and then treatted by CNBr to cut off KSI and brevinin-2CE. The brevinin-2CE waspurified by chromatography on Glutathione Sepharose4B gel peptide, detected by16.5%Tricine-SDS-PAGE electrophoresis. Result showwed the fusion protein was successfully cut intotwo small proteins.Different concentrations brevinin-2CE can completely inhibited the growth of E.coli,produce sharp edges, background completely transparent zone, inhibited the growth ofPseudomonas aeruginosa produces clearer edges, background transparent zone, partially inhibitedthe growth of Staphylococcus aureus produce sharp edges, background deeper zone. The diameterof inhibition zone test showed0.2%brevinin-2CE can be completely inhibited the growth of E.coli, but the1.8%brevinin-2CE enough to completely inhibit the growth of Staphylococcus aureus,brevinin-2CE description of different sensitivity to different pathogensThe antimicrobial peptide brevinin-2CE could exhibitte37.04%inhibitory rate of HCT116 cell when the concentration is100mg/L, and25.93%inhibitory rate of K562. The inhibitory effectwas significantly weaker than the positive control cisplatin. The antimicrobial peptidebrevinin-2CE had no significant inhibitory effect on SMMC27721and A549cells.