Study On Expression Of Design Antimicrobial Peptides In E.Coli And Antimicrobial Activity

Antimicrobial peptides are micromolecular polypeptides which have broad -spectrum antibiosis and generally reside in biosystem. The mechanisms of antibacterial peptides are totally different from antibiotics which inhibit synthesis of microbe's biomacromolecule, so antimicrobial peptide maybe a new antimicrobial agent. Cecropins is the biggest family of antibacterial peptides. It has many good qualities such as wide antimicrobial spectrum, no side effects on animals or plants, and simple structure. In present cecropins'antibiosis mechanism and structure-function relationship know less that restrict the research of high activity antimicrobial peptide by reforming structure and renew design. Prophase of this work had design four antibacterial peptides AMP-A/B/C/D that based on the cecropins'conserved sequence. In this thesis, AMP-A/B/C/D are expressed in Escherichia coli by gene engineering technique, antimicrobial activity of expression production are assayed, finally the structure-function relationship of AMP-A/B/C/D are initially studied by bioinformatics. The work will provide technical data for design and express higher activity antibacterial peptide in future.Engineering bacteria was constructed. Four polypeptides of interest were reverse-translated into DNA sequence based on Escherichia coli bias codons. At aim of getting the AMP-A/B/C/D full gene, seven 50~70bp DNA model-based primer fragments were synthesized by chemical method. PCR amplification respectively ligate fragments and switch in restriction enzyme site (EcoRâ… , Hindâ…¢) by design primer. After reaction of digestion and ligation, the interest gene was cloned into vector PUCI9. Recombinant plasmid PUC-AMP-A/B/C/D was transformed into competent cell using Calcium Chloride. It was confirmed at last that gene of interest had been successfully cloned into PUC19 through DNA sequencing. We successfully get engineering bacteria JM109 -A /B/C/D.Assay of SDS-PAGE gel electrophoresis indicated that the fusion polypeptide was expressed under the induction of IPTG and presented with inclusion body. Engineering bacteria were largely fermented in optimized condition. Fermented strain cells were broken up with ultrasound. Inclusion bodies are in sediment after centrifugal separation. 2M urea washed inclusion body that can remove mostly membrane protein which is in surface of inclusion body. Inclusion body was dissolved using 6M guanidine hydrochloride/2M hydrogen chloride and adding cyanogen bromide to solution Expression production was removed N terminal fusion proteinAMP-A/B/C/D antibacterial activity was detected. The result showed that AMP-A is inactivity, AMP-B/C/D are against Escherichia coli ATCC25922, Salmonella typhi, Bacillus subtilis, Staphylococcus aureus. Minimum inhibitory concentration (MIC) is...