本文利用抗菌肽数据库以及蛋白质分析软件等工具，根据抗菌肽结构与功能的关系，对牛乳铁蛋白肽衍生肽（LfcinBD）的结构进行优化设计。将设计得到的LfcinBD基因片段与pPIC9K质粒构建重组表达载体，线性化后电转化毕赤酵母细胞GS115，利用甲醇诱导表达。实验对发酵上清液进行了分离纯化和活性测试，SDS-PAGE电泳检测到了目标蛋白的有效表达。实验还对优化设计得到的牛乳铁蛋白肽衍生肽与同等发酵条件下产生的天然牛乳铁蛋白肽的抑菌活性进行了对比分析，结果证明该衍生肽对金黄色葡萄球菌有更强的抑菌活性。研究表明经色氨酸Trp替换的牛乳铁蛋白肽中第10，14位氨基酸获得的牛乳铁蛋白肽衍生肽具有很好的抑菌活性，实验为进一步探究牛乳铁蛋白肽衍生肽生物活性的改进奠定了基础。

Antimicrobial peptide databases and protein analysis software were used to optimize the design of bovine lactoferricin-derived peptide (LfcinBD), based on knowledge of the relationship between the structure and function of antimicrobial peptides. The designed lactoferricin-derived peptide gene fragment was inserted into a pPIC9K plasmid to construct a recombinant expression vector. After linearization, Pichia pastoris GS115 cells were transfected by electroporation, and expression was induced with methanol. Isolation, purification, and activity measurements of fermentation supernatants were performed. SDS-PAGE analysis confirmed efficient expression of the target protein. Comparing the optimally designed LfcinBD derivative with natural LfcinB, it was confirmed that the derived peptide had stronger bacteriostatic activity against Staphylococcus aureus. This study indicated that the LfcinB in which amino acid 10 and 14 were replaced with Trp has better antibacterial activity. This work lays the foundation for further exploration to improve the biological activities of LfcinBD.

江苏高校优势学科建设工程资助项目；四川省科研院所科技成果转化资金项目(15010116)；江苏大学高级专业人才科研启动资金（12JDG069）