Construction Of Tandem Expression Vector Expressing Antibacterial Peptide Dermadistinctin And Identification Of Antimicrobial Activity Of The Expressed Fusion Protein

Nowday,resistant strains have evolved,as they will almost inevitably do given sufficient time of exposure. A viable solution to this acquired resistance problemmay reside in new antibiotics,dissimilar to those that have lost their effectiveness. And the emergence of antimicrobial peptides undoubtedly troubled anti-infective treatment sheds some light. Antibacterial peptides (AMPs)include a series of small peptides with positive ions most of which have abroad-spectrum antibiotic and they are significant ingredients during the course of congenital immunity in most creatures. Antibacterial peptides will not induce the resistance of bacteria because of its unique antibacterial machnism, which suggests that AMPs can be a potential substitute of traditional antibiotics.There are two sources for AMPs:isolating from the skin-secretory glands of the frog Phyllomedusa distincta. Many of these peptides were described under the name of dermaseptins. They all showed potent antimicrobial activity against Gram-negative and Gram-positive bacteria, especially resistant Staphylococcus aureus demonstrated effective antimicrobial, antibiotic-resistant bacteria strains present in large numbers, the development of antimicrobial peptides DDK and DDL have the potential conditions to become the new of antimicrobial agents.But according to our knowledge,there is no report on expression of Dermadistinctin gene in any type of expression system. The purpose of this research is to develop recombinant antibacterial peptide Dermadistinctin through the technique of gene engineering and protein engineering and to study the in vitro and in vivo activity, which lay a foundation for the mass production of AMPs and the safeguarding of animal husbandry. The study was conducted from following aspects:1 Based on the gene sequences encoding antibacterial peptide DDK,DDL registered in GenBank, DDK and DDL genes with enterokinase cleavage sites were designed and synthesized respectively according to the preferential condon of E.coli by SOE method,Length were 148 bp and 121bp. 2 The modified antibacterial peptide gene DDK and DDL were both cloned into the pET-32a to construct the recombinant expression vector P-DDKL which was subsequently transferred into Rosetta(DE3) and induced under IPTG.3 Different conditions were detected to adjust the expression of DDK and DDL Fusion expressional protein of DDK and DDL was obtained, and the maximum expressional production was achieved after inducting by 0.1 mM IPTG for 4 hours.4 Then the fusion protein named DDKL was purified, and cleaved by enterokinase to generate antibacterial peptides DDK and DDL. According to MIC and MBC,the recombinant had abroad-spectrum antibacterial activity on both gram-postive,esp-ecially showed poptent antibacterial activity against ampicillin-resistant bacteria.5 Kunming mouse were used to study the elementary effectiveness of preventing and treating bacterial infection.In the preliminary experiment, the MLD100 of Escherichia coil O1 to mouse were determined. The mice with celiac injection of 200μg purified recombinant antibacterial peptide were detected alive in 48 hours after infection with MLD100 of Escherichia coil O1.