Construction And Expression Activity Of Novel Fusion Gene Of Antibacterial Peptides

In order to construct a fusion protein which possibly has broad-spectrum and efficacious antibacterial activities, or could increase or enhance one or more new biologic activities to strengthen animal defense against pathogen invasions, we synthesized the fusion gene combinating three antibacterial peptides(ESC, citropin1.1 and DHV4,which were ESC, CIT1 and DHV4 as short term) using the overlap extension PCR technique. Two flexible linker peptides(Gly+Gly+Gly+Ser+Gly+Gly+Ser and Gly+Gly+Ser+Gly+Gly+Ser+Gly) consisted of serine and glycine were inserted into the fusion peptide so as to ensure no disturbance among their activity domains followed with analysis and adjustment by the software of ANTHEPROT 5.0 on its three-dimension structure and amino acid sequence.Bias on some codens in fusion gene when expressing in E.coli were considered, and then some synonymous mutations were done and four oligodeoxynucleotide fragments less than 80bp were designed and artificially synthesized with the help of highly fidelity of Pfu DNA polymerase.Prokaryotic expression vector of pGEX-4T-1-ESC/CIT1/DHV4(pG-ECD as short form) was constructed successfully for the first time after being digested by double enzymes of BamH I/EcoR I, plasmid PCR and sequence identification. Then recombinant plasmids were transformed into E.coli DH5α and BL21(DE3), expressed in 2× YTA culture medium with IPTG, followed by SDS-PAGE to analyze its property. The result showed that the 33kD of molecular weight was consistent...