石斑鱼-防御素的酵母表达及其产物抗菌活性分析

防御素是一类阳离子抗菌肽。研究从石斑鱼垂体SMART cDNA 文库中扩增出129 bp 石斑鱼-防御素成熟肽序列, 将其克隆到毕赤酵母表达载体pPCIZA 中, 构建了石斑鱼-防御素的真核表达载体, 电击转化毕赤酵母GS115。Western Blot 分析表明石斑鱼-防御素在酵母菌中获得了表达。体外抗菌实验表明纯化的重组蛋白具有抑制大肠杆菌以及嗜水气单胞菌的作用, 但是对革兰氏阳性菌, 如金黄色葡萄球菌和藤黄微球菌的生长没有抑制作用。实验结果表明酵母表达的石斑鱼-防御素能够特异地抑制革兰氏阴性菌的生长。  

THE YEAST EXPRESSION OF GROUPER -DEFENSIN AND ANTIBACTERIAL ACTIVITY OF THE RECOMBINANT PROTEIN

Defensin, a small cationic peptide, is an antimicrobial peptide, exhibit broad-spectrum antibacterial activity. They spread widely in plants, invertebrate and vertebrate animals, which can rapidly kill bacteria, fungi and viruses etc. Bacteria and viruses limited the production of grouper (Epinephelus coioides). In this study, we cloned grouper -defensin open reading frame, to express in the Pichia pastoris, and detect the bioactive of recombinant protein against bacteria. First, grouper -defensin cDNA was amplified by polymerase chain reaction (PCR) from pituitary cDNA library of a protogynous hermaphroditic orange-spotted grouper. The 129 bp DNA fragment encoding mature grouper -defensin peptide was subcloned into vector PMD18-T, then inserted into the yeast expression vector pPCIZA and transfected into Pichia pastoris GS115 expression host by electroporation. The genome of Pichia pastoris clone was extracted as template for screened the positive clone by using PCR. After grouper -defensin was secreted by GS 115 by small-scale culture, the recombinant grouper -defensin was induced by methanol for large scale. The supernatant was collected in 24h, 48h and 72h post induction for the recombinant protein detection. Subsequently, the grouper -defensin peptide was detected in the supernatant of transfected yeast by Western Blot analysis. The purified recombinant grouper -defensin crude extract from transfected P. pastoris showed antimicrobial activities against Escherichia coli and Aeromonas hydrophila. However, recombinant protein did not inhibit the growth of Gram-positive bacteria, such as Staphylococcus aureus and Micrococcus luteus. Bioactivity analysis indicated that the antibacterial activity of the recombinant grouper -defensin expressed in P. pastoris specific to Gram-negative bacteria. Grouper -defensin have antibacterial activity-specific to Gram-negative bacteria, this specificity is in correspondence with the environment of grouper.