重组牛乳铁蛋白肽衍生肽设计及其在毕赤酵母中的表达

Design of a recombinant bovine lactoferricin peptide and its expression in Pichia pastoris

利用抗菌肽数据库和生物信息学分析工具,基于抗菌肽结构和功能的关系,设计了LFcinB衍生肽LFcinB-W4,10。为了验证衍生肽设计的合理性及表达产物功能正确性,将优化设计的基因序列经限制性内切酶酶切后,用T4DNA连接酶连接到分泌型表达载体pPIC9K中,获得的重组表达载体pPIC9K-LFcinB-W4,10经线性化后电转入毕赤酵母GS115中,经过G418浓度梯度筛选得到高拷贝转化子。经PCR鉴定,目的基因与酵母基因组稳定整合。阳性转化子经体积分数为2.5%的甲醇诱导表达,表达产物用Tricine-SDS-PAGE检测到相对分子质量约为3.2kDa的衍生肽LFcinB-W4,10。每24h取样检测发酵上清液抑菌活性,结果表明,抗菌肽LFcinB-W4,10对金黄色葡萄球菌具有良好的抑菌活性,诱导表达96h时其抑菌圈直径最大。实验为探究牛乳铁蛋白肽功能-结构关系奠定了基础。

A LfcinB-derived peptide LfcinB-W4,10 was designed based on the structure-function relationship of antimicrobial peptides based on antimicrobial peptide database and bioinformatics analysis. In order to verify the rationality of the designed derivative peptide and the functional correctness of the expression product, the optimized gene sequence was digested with restriction endonucleases and ligated into the secretory expression vector p PIC9K with T4 DNA ligase. The recombinant expression vector p PIC9K-LFcinB-W4,10 was linearized by Sac I and transformed into Pichia pastoris GS115 by electroporation, and G418 was used to obtain high copy transformants. The results showed the target gene was stably integrated with the yeast genome. The expression of positive transformants were induced by2.5%( v/v ) methanol, and the expressed product was LfcinB-W4,10 with a relative molecular weight of3.2 kDa detected by Tricine-SDS- PAGE. Moreover, the antibacterial activity of the fermentation supernatant was measured every24 h. The results showed the derived peptide LfcinB-W4,10 had good antibacterial activity against Staphylococcus aureus , and the bacteriostatic ring diameter was the largest after 96 h induction. In conclusion, this study lays a foundation to further explore the relationship between function and structure of bovine lactoferrin peptides.