Design Of Antimicrobial Peptide Based On Cecropins And Its Genetic Expression

Characterized by wide antimicrobial spectrum, high antimicrobial activities, less possiblity to produce drug tolerance and no side effects on animals or plants, antimicrobial peptide (AMP) as ideal substitute for antibiotics has aroused people's more and more interests. Cecropin belongs to one of AMP's families in terms of taxon. In recent years, much more lead AMPs were designed by amino acid replacement or sequence recombination based on knowledge of cecropins. But this method brought out the drawback by which it was tolerated to the chance on the discovery of lead AMP. A research based on cecropin's structure-activity relationship was conducted as follows: many consensus fragments (of cecropins) being pieced together into new AMPs. The designed AMP's genetic expression and antimicrobial activities were investigated.Sixty-two cecropin AMPs were screeningly accumulated against the database of Swiss-Prot by utilizing sequence retrieval system (SRS). They were divided into 11 groups according to their sources and consequently, multiple consensus sequences were found by method of pattern discovery. Various consensus sequences were pieced together into many different polypeptides according with the criterion of 60-amino-acid model based on substantial recombinant rules. Analysis was applied to each polypeptide on its basic physico-chemical characters, transmembrane condition, distribution of electronic fields and statistical models. As a result, a 61-amino-acid polypeptide was designed and treated as the expression polypeptide of interest. Polypeptide of interest was converted into 235 bp gene tailored to Escherichia coli bias codons. At aim of getting the full 235 bp gene, five 50~70 bp model-based primers were first synthesized by chemical method followed by 3-step PCR amplification in an effort to ligate them into full gene. Expression vector pUC19_Cec was constructed. It was confirmed by agarose gel electrophoresis that there was a similar band with gene of interest; genetic sequencing had also validated the consistency of experimental sequence with gene of interest. Genetically-engineered strain was fermented and its expressed products were isolated by SDS-PAGE gel electrophoresis which presented with the same band as polypeptide of interest. The amino acid composition of expressed protein was consistent with polypeptide of interest through high-performance chromatography analysis; As a result, polypeptide of interest was successfully expressed in E.Coli JM109. Polypeptide of interest was expressed under the induction of IPTG during fermentation and presented with inclusion body (IB) intracellully. Fermented strain cells were broken up with the aid of ultrasound. IBs were dissolved by using low concentration urea in order to retain polypeptide of interest. Optimized process for dissolving IBs was determined as follows: at the temperature of 4 ℃, ultrasound interval being 6 s, washing concentration of urea being 2.0 mol / L, dissolving concentr- ation of urea being 4.0 mol / L.Assay of bioactivities for expression polypeptide was studied and it indicated that expression polypeptide had biological resistance against Escherichia coli ATCC25922, Salmonella typhi, Bacillus subtilis, Staphylococcus aureus, Bacillus thuringiensis, Bacillus acidi lactici and Lactic acid galactococcus. The maximum and the minimum inhibitory zone were 11.3mm and 6.5mm for Staphylococcus aureus and Salmonella typhi respectively. Minimum inhibitory concentration (MIC) trial for strains mentioned above indicated that the maximum MIC was 12.1 mg / L for Staphylcoc- cus aureus and the minimum MIC was 25.6 mg / L for Salmonella typhi. MICs for other strains were between those of the previous two strains.