Optimization of the expression and biological characteristic of antibacterial peptide Fowlicidin-3 and its therapeutic tests on Escherichia coli O1 infected chickens | 鸡源抗菌肽Fowlicidin-3表达条件的优化及生物学特性和体内疗效研究

chinois. 摘　　要：对重组X-33毕赤酵母菌株表达的鸡源Fowlicidin-3抗菌肽的条件进行优化,并研究了表达产物的生物学活性。结果表明：当含有重组酵母菌的BMGY培养基A600值达到5～6时,转接pH 6.0的BMMY培养基,每24 h补加2%甲醇,29℃条件下培养72 h,重组鸡源Fowlicidin-3抗菌肽获得高效表达,含量达到170 mg.L-1。采用琼脂孔扩散法对该抗菌肽的热稳定性、酸碱稳定性等特性进行分析,结果表明：鸡源Fowlicidin-3抗菌肽具有较强的热、酸稳定性;对致病性大肠杆菌K99、鸡白痢沙门氏菌和金黄色葡萄球菌CowanⅠ的最小抑菌浓度分别为3.12、1.56和1.56μg.mL-1。在对大肠杆菌O1感染雏鸡的体内疗效试验中,鸡源Fowlicidin-3显示出了较好的预防和治疗效果。

anglais. The requirement for the flask-shaking culture fermentation of the recombinant antibacterial peptide Pichia pastoris was optimized.Fowlicidin-3 would be expressed with high level and the concentration of the secreted peptides was 170 mg・L-1 while the density of the yeast amounted to A600 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.According to Agarose Diffusion Assay,the pH-stable assay indicated that the peptide dispalyed the best activities while the pH was between 5.0-10.0.The heat-stable assay indicated that the peptide could remain its inhibition activity after 2 h treated in boiled water.The minimal inhibitory concentration（MIC）of antibacterial peptide Fowlicidin-3 against E.coli K99,Salmonella typhimurium and Staphylococcus aureus CowanⅠwere 3.12,1.56 and 1.56 μg・mL-1,respectively.The results of therapeutic tests showed that the mortality rate decreased significantly（P〈0.01）in both antimicrobial peptide Fowlicidin-3 group and ciprofloxacin group compared to the E.coli O1 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 displayed preferable curative effect and protection.