水生生物学报2011，Vol.35Issue(5)：739-744,6.DOI:10.3724/SP.J.1035.2011.00739
石斑鱼β-防御素的酵母表达及其产物抗菌活性分析
THE YEAST EXPRESSION OF GROUPER β-DEFENSIN AND ANTIBACTERIAL ACTIVITY OF THE RECOMBINANT PROTEIN
摘要
Abstract
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 P-defensin open reading frame, to express in the Pichia pastoris, and detect the bioactive of recombinant protein against bacteria. First, grouper P-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 P-defensin peptide was subcloned into vector PMD18-T, then inserted into the yeast expression vector pPClZaA 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 p-defensin was secreted by GS 115 by small-scale culture, the recombinant grouper P-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 p-defensin peptide was detected in the supernatant of transfected yeast by Western Blot analysis. The purified recombinant grouper P-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. Bioactiviry analysis indicated that the antibacterial activity of the recombinant grouper p-defensin expressed in P. Pastoris specific to Gram-negative bacteria. Grouper p-defensin have antibacterial activity-specific to Gram-negative bacteria, this specificity is in correspondence with the environment of grouper.关键词
石斑鱼β-防御素/酵母表达/抗菌活性Key words
Grouper p-defensin/Yeast expression/Antibacterial activity分类
生物科学引用本文复制引用
金俊琰,周莉,桂建芳..石斑鱼β-防御素的酵母表达及其产物抗菌活性分析[J].水生生物学报,2011,35(5):739-744,6.基金项目
国家自然科学基金项目(U0631007) （U0631007）
973项目(2010CB126301) （2010CB126301）
淡水生态与生物技术国家重点实验室创新项目(085B181F01)资助 （085B181F01）
