众所周知，抗生素作为治疗病原感染的有力武器，在人类医疗领域以及畜牧业中都占有重要的地位。但是，抗生素的滥用也给病原菌和环境微生物造成很大选择压力，并由此而诱导产生出具有多种抗生素抗性的微生物菌株，同时由于新型抗生素的开发很耗时，抗生素的应用往往也无法跟上耐药菌株产生的速度，给人类以及畜禽健康带来严重危机，这也使得人们对安全、有效的新型抗菌制剂的寻求更为迫切。抗菌肽作为动物免疫防御系统的一个重要组成部分，这类多肽具有杀菌谱广、作用机制独特、热稳定性高等优点。鉴于以上优点，抗菌肽成为具有巨大发展潜力的新型抗菌药物。本研究的目的就是应用基因工程和蛋白质工程技术研制出鸡源Fowlicidin-3重组抗菌肽，并研究其体内外抗菌活性，为重组抗菌肽的工程化生产及其在临床上的应用奠定基础。本研究主要从以下几个方面展开。 1 根据抗菌肽数据库中Fowlicidin-3的氨基酸序列，选用毕赤酵母的偏嗜密码子，设计抗菌肽的基因，通过SOE法合成得到全长为114 bp的Fowlicidin-3基因。将Fowlicidin-3基因克隆入pPICZα-A质粒，构建分泌型重组酵母表达载体pPICZα-A-F。将线性化重组表达载体pPICZα-A-F转入毕赤酵母宿主菌X-33中，获得分泌表达量达到150 mg/L的Fowlicidin-3多肽，对致病性大肠杆菌K99和鸡白痢沙门氏菌显示出较好的抑制活性，抑菌圈直径分别为1.9 cm和2.2 cm。 2 本试验选用X-33毕赤酵母菌株表达鸡源Fowlicidin-3抗菌肽，对表达条件进行优化并进行表达产物的生物学活性研究。结果表明，当含有重组酵母菌的BMGY培养基的OD600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后，鸡源Fowlicidin-3抗菌肽获得高效表达，含量达到170 mg/L。采用琼脂孔扩散法对该抗菌肽的热稳定性、酸碱稳定性等生物学活性进行分析，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、鸡白痢沙门氏菌和金黄色葡萄球菌的最小抑菌浓度分别达到3.12 µg/mL、1.56 µg/mL和1.56 µg/mL。 3 为比较研究重组Fowlicidin-3抗菌肽对大肠杆菌感染鸡的体外疗效，本试验以21日龄AA肉雏鸡作为研究对象，利用重组Fowlicidin-3抗菌肽对大肠杆菌感染雏鸡进行体外保护及治疗试验。试验结果显示，保护性实验中，Fowlicidin-3上清组、环丙沙星组与感染对照组相比，死亡率显著降低，且Fowlicidin-3上清组保护与防病效果与环丙沙星效果相当；治疗性实验中，抗菌肽上清治疗组、环丙沙星治疗组与感染对照组相比死亡率显著降低，并且抗菌肽Fowlicidin-3上清组对大肠杆菌病有较好的体内治疗效果，与抗生素环丙沙星显示的效果相当。

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 pPICZα-A vector to construct the recombinant expression vector pPICZα-A-F. The linearized plasmid pPICZα-A-F was transformed into P. pastoris X-33 by electroporation. Under the control of the promoter AOX1(alcohol oxidase1), 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 (CowanⅠ) were 3.12 µg/mL,1.56 µg/mL,1.56 µg/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 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 has preferable curative effect and protection.