| 摘要: |
| 从实验室保存的pBS-Trx重组质粒中克隆球石藻病毒EhV-Trx基因, 构建毕赤酵母重组表达载体pPIC9K-EhV-Trx, 将重组质粒电转化毕赤酵母GS115, 诱导分泌表达并对重组蛋白进行二硫键还原酶活性分析。结果表明, EhV-Trx 基因开放阅读框为591bp, 编码197个氨基酸; 在毕赤酵母GS115中成功诱导表达重组EhV-Trx, 经SDS-PAGE 分析目的蛋白分子量约为27.8kDa; 重组EhV-Trx具有二硫键还原酶的活性, 能有效打开胰岛素A、B两条链的二硫键, 有望开发成一种新型的硫氧还蛋白脱敏制剂应用于食品安全领域。 |
| 关键词: 海洋球石藻病毒(EhV), 硫氧还蛋白, 毕赤酵母, 分泌表达, 活性分析 |
| DOI：10.11693/hyhz201205005005 |
| 分类号: |
| 基金项目:福建省自然科学基金项目, 2010J01261 号; 集美大学中青年创新团队专项基金资助, 2010A007 号 |
附件 |
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| EXPRESSION AND ACTIVITY ANALYSIS OF THIOREDOXIN (Trx) FROM MARINE COCCOLITHOPHORID EMILIANIA HUXLEYI VIRUS IN PICHIA PASTORIS |
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CAI Yi-Qin, ZHANG Zhi-Lan, LUO Bang-Bin, LIU Jing-Wen
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College of Bio-Engineering, Jimei University
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| Abstract: |
| Thioredoxin (Trx), a small molecule ubiquitous multifunctional acidic protein, was found to reduce disulfide bonds of protein (convert SOS to 2 SH) and thereby mitigate the allergenicity of food. Emiliania huxleyi virus (EhV) is the first virus containing Trx gene reported. Here the gene of EhV-Trx was amplified from the recombinant plasid of pBS-EhV99B1-Trx by PCR. Ehv-Trx gene was inserted yeast expression vector pPIC9K to construct recombinant expression plasmid pPIC9K-EhV99B1-Trx. After sequencing, the recombinant expression plasmid was transformed into Pichia pastoris GS115 by electroporation method. The recombinant protein EhV99B1-Trx reduction activity was detected. The results showed that: the open-reading frame (ORF) of EhV99B1-Trx encoded a protein of 197 amino acids; the recombinant EhV99B1-Trx was successfully induced expression in P. pastoris GS115 and the target protein molecular mass was about 27.8kDa; as found for other proteins with intramolecular disulfide bonds, insulin were reduced specifically by the recombinant EhV99B1-Trx and the treated product was relatively stable, which indicated that the EhV99B1-Trx, as a new kind of thioredoxin, do have the potential in food safety areas. |
| Key words: Emiliania huxleyi virus (EhV), Thioredoxin (Trx), Pichia pastoris, Secreted expression, Activity analysis |
