| 摘要: |
| 通过PCR 技术从副溶血弧菌(Vibrio Parahaemolyticus, VP)基因组DNA 上扩增耐热溶血素基因tdh, 双酶切后定向插入到酵母表达载体pPICZaA 中, 构建重组表达质粒pPICZaA-tdh, 经限制性内切酶SacI 线性化后电转化毕赤酵母(Pichia pastoris, P. pastoris)GS115, 经PCR 和测序鉴定耐热溶血素基因成功整合到毕赤酵母的基因组中。在甲醇诱导下进行 tdh 的表达, SDS-PAGE 分析证明重组 TDH(thermostable direct hemolysin, TDH) 的分子质量约为23 ku, 经 Western blotting 鉴定VP 抗体能与表达的 TDH 发生特异反应, 说明 tdh 基因在毕赤酵母中的表达是准确的。溶血实验证明了表达的TDH 具有溶血活性。本研究首次应用毕赤酵母表达耐热溶血素基因 tdh, 在培养基中获得分泌表达的耐热溶血素, 为研究tdh 基因的功能奠定了基础。 |
| 关键词: 耐热溶血素 分泌表达 毕赤酵母 溶血活性 |
| DOI： |
| 分类号: |
| 基金项目:国家863 计划项目(2006AA100306; 2006AA100307) |
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| Expressing the tdh gene of Vibrio parahaemolyticus in Pichia pastoris and its hemolysis activity |
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ZHAO Yong-gang,ZHAN Wen-bin,SAREN Gao-wa,WANG Jun-wei,WANG Zhi-liang
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| Abstract: |
| We cloned thenmostable direct hemolysin gene (tdh) from the genome DNA of Vibrio parahaemolyticus polymerase chain reaction (PCR). The gene was ligated into eukaryotic expression vector pPICZaA. The recombinant plasmids pPICZaA-tdh was transformed into P. pastoris GS115 cells by electroporation after SacI digestion. After the transformed cell was induced with methanol, one protein of 23 kDa appeared in the medium as detected by SDS PAGE. Western blotting showed that recombinant TDH would react with rabbit antiserum immunitied by VP antigen. The recombinant protein also showed hemolytic activity. This is the first report describing successful expression of the tdh gene in P. pastoris and consequent production of secretory recombinant TDH, providing a framework for further structural and functional studies. |
| Key words: Thenmostable direct hemolysin Secretory expression Pichia pastoris Hemolysis |
