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。
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-EhV99B 1-Trx by PCR. Ehv-Trx gene was inserted yeast expression vector pPIC9K to construct recombinant expres- sion plasmid pPIC9K-EhV99B1-Trx. After sequencing, the recombinant expression plasmid was transformed into Pichia pastoris GSl15 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 recombi- nant EhV99B l-Trx was successfully induced expression in P. pastoris GS 115 and the target protein molecular mass was about 27.SkDa; 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.
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2012年第5期905-910,共6页
Oceanologia Et Limnologia Sinica
基金
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
