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Scygonadin抗菌肽是一类阴离子抗菌肽,从锯缘青蟹体内分离得到,对溶壁微球菌和嗜水气单胞菌具有较好的抑制作用。研究从锯缘青蟹c DNA文库中找出Scygonadin抗菌肽的成熟核苷酸序列和对应的氨基酸序列,并根据毕赤酵母的偏好密码子进行密码子优化,优化后的密码子经人工合成后连接到毕赤酵母表达载体p PCIZαA上,构建锯缘青蟹Scygonadin抗菌肽的真核表达载体,然后经电击转化至毕赤酵母X-33中。表达产物经镍柱纯化后进行SDS-PAGE电泳。结果表明,锯缘青蟹Scygonadin抗菌肽在毕赤酵母X-33中获得表达,且测序结果显示与最初设计的氨基酸序列一致;体外抑菌试验表明:纯化的重组多肽具有抑制溶壁微球菌及嗜水气单胞菌的作用,而且还可抑制金黄色葡萄球菌等多种革兰阳性菌,但其对革兰阴性菌的抑制作用相对较弱。
Scygonadin antimicrobial peptide is an anionic antimicrobial peptide, isolated from the body of Merlin, and has a good inhibitory effect on Micrococcus furiosus and Aeromonas hydrophila. In this study, the mature nucleotide sequence and corresponding amino acid sequence of Scyngadadin antimicrobial peptide were found from c DNA library of P. mandarina, and codon optimized based on the preferred codon of Pichia pastoris. After the optimized codon was synthesized Was ligated into the Pichia pastoris expression vector p PCIZαA to construct an eukaryotic expression vector for Scygonadin antimicrobial peptides, and then transformed into Pichia pastoris X-33 by electroporation. The expressed product was purified by nickel column and subjected to SDS-PAGE electrophoresis. The results showed that the Scygonadin antimicrobial peptide was expressed in Pichia pastoris X-33, and the sequence was consistent with the originally designed amino acid sequence. The antibacterial activity in vitro showed that the purified recombinant peptide could inhibit Micrococcus lysostigma and Aeromonas hydrophila, but also can inhibit Staphylococcus aureus and other Gram-positive bacteria, but its inhibition of Gram-negative bacteria is relatively weak.