Fusion Expressing Of Magainin And Thanatin Hybrid Antibacterial Peptide In Escherichia Coli

Antimicrobial peptide,as a major component in biological immune system,belonged to a class of amphiphilic micro-molecule polypeptide with positive charge.It was produced by organism in the process of immune response and was widely applied to medicine,food and animal husbandry.A new hybrid peptide produced from two or more antimicrobial peptide fragments could offset the disadvantages of natural antimicrobial peptides and increase its antibacterial activity.In this study,the active site of Magainin and Thanatin antimicrobial peptides were spliced and a novel hybrid peptide MT were then obtained.The nucleotide sequence of the hybrid peptide was designed and analyzed based on the sequence of the MT gene and the codon preference of E.coli.Three primers were designed by SOE-PCR technology,the target MT gene was obtained by two rounds of PCR amplification,the target gene was ligated into vector pGEX-6P-1,the expression plasmid pGEX-6P-1-MT was construct to express the target gene;recombinant expression vector was then transformed into the expression strain E.coli BL21(DE3),and the genetically engineered strain pGEX-6P-1-MT/BL21(DE3)was constructed.The fusion protein was purified by GST agarose gel column after optimizing the fermentation conditions,including IPTG concentration,induction time,temperature and medium.The antibacterial activity of the hybrid antimicrobial peptides was analyzed after the cleavage of the enterokinase.The main results of this paper are as follows:(1)The Bioinformatic prediction based on physicochemical parameters,secondary structure and hydrophilicity of the MT showed that the length of the MT peptide was 29 amino acids,its molecular weight was 3.35 kDa and its isoelectric point of pI was 10.57.The hybrid peptide contained alpha helix and beta fold structure.The results of the hydrophilic mapping showed that the hybrid peptide was amphiphilic and might have antibacterial activity.According to the codon preference of E.coli,the gene sequence was optimized and the target gene was obtained.(2)The genes of the hybrid antimicrobial peptide MT were synthesized by two rounds of PCR using the overlapping three-step primer(SOE-PCR).(3)The target gene MT and expression vector pGEX-6P-1 were digested by Eco R I and BamH I,and the recombinant expression vector pGEX-6P-1-MT is successfully constructed.(4)SDS-PAGE was performed after sonication.The results showed that the fusion protein was expressed in soluble form in the cell supernatant.Western-blot results showed that the target protein was expressed.The target protein was then purified by GST agarose gel purification column.The fusion protein was obtained,accounting for 50.1% of the total bacterial cells,49.5 mg/L protein,and the target protein with molecular weight of 3.35 k Da was obtained after intestinal kinase cleavage.(5)Through the optimization of the induced expression conditions of the hybrid antimicrobial peptide MT recombinant bacteria,the optimized conditions of the MT was obtained: IPTG concentration of 0.8 mM;induction time of 3 h;induction temperature of 37? and TB medium.(6)The antimicrobial activity of hybrid antimicrobial peptide MT was studied by inhibition zone method.The results showed that the heterozygous peptide had inhibitory effect on E.coli DH5?,Staphylococcus aureus and Bacillus subtilis,and the size of inhibition zone were 5 mm,6 mm and 10 mm,the inhibitory effect on Bacillus subtilis was stronger than that of the former two.The minimum inhibitory concentrations were 20 ?M,16.5 ?M,9 ?M,respectively.In this study,the soluble expression of hybrid peptide MT in E.coli pGEX-6P-1 expression vector was successfully achieved,and the MT-target protein with high purity and antibacterial activity was obtained by purification.It will have a certain value.