| As is known, antibiotics is an effective weapon to against pathogenic microorganisms both in the field of human and livestock pharmacy. However, selection pressure to pathogen and environmental microorganisms are caused by abusing of antibiotics, which also induced many mul-antibiotic-resistant bacterium. The time-consuming exploitation of new types of antibiotics and the rapider emergence of antibiotic-resistance bacterium lead to spread of diseases. To find safety, effective antibacterial agents are considerable importance. As an important component of innate immunological system, antibacterial peptides have many advantages:wide antibiotic spectrum, high sensitive to bacterials, unique functional mechanism, high heat-stability, no toxicty, no remnant, which show a huge potential to be a new antibacterial agents. The purpose of this study is to develop recombinant ABP Fowlicidin-3 through the technique of gene engineering and protein engineering with the property right reserved and investigate the activity in vitro and in vivo, which supply groundwork for the recombinant ABP manufacture production and application in clinic of livestock. The study includes the following aspects.1 According to the amino acid sequences of Fowlicidin-3 as described in the antibacterial peptide database, using the preferential condon of P. pastoris, the antibacterial peptide Fowlicidin-3 gene 114 bp in length was designed and synthesized by SOE PCR. The antibacterial peptide gene was cloned into the pPICZa-A vector to construct the recombinant expression vector pPICZa-A-F. The linearized plasmid pPICZa-A-F was transformed into P. pastoris X-33 by electroporation. Under the control of the promoter AOX1(alcohol oxidasel), Fowlicidin-3 was expressed and secreted into supernatant. The concentration of the secreted peptides was 150 mg/L. Agrose diffusion assay showed that Fowlicidin-3 had strong antibacterial abilities to E. coli K99 and Salmonella pullorum, the diameter of inhibition zone to E. coli K99 and Salmonella pullorum were 1.9 cm and 2.2 cm, respectively.2 The requirement for the flask-shaking culture fermentation of the recombinant antibacterial peptide P. pastoris was optimized. It was found that the recombinant Fowlicidin-3 would be expressed with high level while the density of the yeast amounted to OD600 value 5-6, the pH of the culturing BMMY medium was 6.0, the yeast was cultured under 29℃for 72 hours and 2% methanol was supplemented into the medium, The concentration of the secreted peptides was 170 mg/L.. According to Agarose Diffusion Assay, the heat-stable and pH-stable characteristics of Fowlicidin-3 peptide were examined. The pH-stable assay indicated that the peptide dispalyed the best activities while the pH was 5.0-10.0, and pH 2.0-12.0 still had certain activities. The heat-stable assay indicated that the peptide could remain its inhibition activity after being treated for more than 2 hours in boiled water. The Minimal inhibitory concentration (MIC) of antibacterial peptide Fowlicidin-3 against E.coli k99, S. typhimurium and S. aureus (Cowan I) were 3.12μg/mL,1.56μg/mL,1.56μg/mL, respectively.3 To compare the effects of recombinant antibacterial peptide Fowlicidin-3 on the bacterial infected chickens in vivo, therapeutic and protective tests of recombinant antibacterial peptide Fowlicidin-3 in the E.coli O1 challenged twenty-one days old chickens were studied. The results showed that the mortality rate decreased significantly in both antimicrobial peptide Fowlicidin-3 group and ciprofloxacin group compared to the E.coli 01 infection control group, and the prevention effectives from antibacterial peptide Fowlicidin-3 group reached the high level as ciprofloxacin group did. Recombinant antibacterial peptide Fowlicidin-3 has preferable curative effect and protection. |
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