School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China.
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China.
Biochem Biophys Res Commun. 2021 Jan 1;534:822-829. doi: 10.1016/j.bbrc.2020.10.098. Epub 2020 Nov 22.
Bovine lactoferrin peptide has been shown to be a broad-spectrum antimicrobial peptide. Based on the relationship between the structure and function of antimicrobial peptides, the antimicrobial peptide databases and protein analysis software were used to optimize the design of bovine lactoferricin peptide (LfcinB). The designed bovine lactoferricin-derived peptide (LfcinBD) gene fragment was inserted into a pPIC9K-His plasmid to construct a recombinant expression vector. After linearization of the Recombinant plasmid, Pichia pastoris GS115 cells were transfected with linearized recombinant plasmid by using electroporation and LfcinBD gene expression was induced with methanol. After the fermentation, supernatant was separated by low-temperature high-speed centrifugation. Ultrafiltration and freeze drying of the fermentation supernatant were performed, purified. Experimental results showed that the LfcinBD had stronger bacteriostatic activity against Staphylococcus aureus than the natural bovine lactoferrin peptide (LfcinB) produced under the same fermentation conditions. The effective expression of the optimized bovine lactoferricin-derived peptide was detected using SDS-PAGE electrophoresis. This study lays the foundation for further exploration to improve the biological activities of antimicrobial peptides.
牛乳铁蛋白肽已被证明是一种广谱抗菌肽。基于抗菌肽结构与功能的关系，利用抗菌肽数据库和蛋白质分析软件对牛乳铁肽（LfcinB）进行优化设计。设计的牛乳铁蛋白衍生肽（LfcinBD）基因片段插入到 pPIC9K-His 质粒中，构建重组表达载体。线性化重组质粒后，用电穿孔法将线性化的重组质粒转染毕赤酵母 GS115 细胞，用甲醇诱导 LfcinBD 基因表达。发酵结束后，采用低温高速离心分离上清液。对发酵上清液进行超滤和冷冻干燥，进行纯化。实验结果表明，LfcinBD 对金黄色葡萄球菌的抑菌活性强于在相同发酵条件下产生的天然牛乳铁蛋白肽（LfcinB）。SDS-PAGE 电泳检测到优化后的牛乳铁蛋白衍生肽的有效表达。本研究为进一步提高抗菌肽的生物活性奠定了基础。
