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目的在毕赤酵母中分泌表达细粒棘球绦虫硫氧还蛋白过氧化物酶(Echinococcus granulosus thioredoxin peroxidase,EgTPx),并检测其抗氧化活性。方法从包囊中分离原头蚴,抽提总RNA,采用RT-PCR获取EgTPx的cDNA片段并克隆至分泌型表达载体pPIC9K,构建重组表达质粒pPIC9K-EgTPx,电穿孔法转化毕赤酵母菌株GSll5,在MD平板上筛选His+克隆,采用梯度浓度G418抗性筛选高拷贝转化子,提取酵母基因组DNA,采用PCR鉴定Mut表型,阳性克隆经甲醇诱导表达EgTPx蛋白,表达产物经SDS-PAGE和Western blot鉴定,并检测其抗氧化活性。结果酶切和测序证实重组表达质粒pPIC9K-EgTPx构建正确,表达的目的蛋白分子质量单位约为22ku,该蛋白可被鼠抗EgTPx多抗识别,且具有较强的抗氧化活性。结论在毕赤酵母表达系统中成功表达了EgTPx,该蛋白具有免疫反应性和抗氧化活性。
Objective To excrete and express Echinococcus granulosus thioredoxin peroxidase (Echinococcus granulosus thioredoxin peroxidase, EgTPx) in Pichia pastoris and test its antioxidant activity. Methods Total RNA was isolated from the cysts and RNA was extracted. The cDNA fragment of EgTPx was obtained by RT-PCR and cloned into secretory expression vector pPIC9K. The recombinant plasmid pPIC9K-EgTPx was constructed and electroporated into Pichia pastoris GS115 , And His + clones were screened on MD plates. High-copy transformants were screened by gradient G418 resistance. The yeast genomic DNA was extracted. Mut phenotypes were identified by PCR. The positive clones were induced by methanol to express EgTPx protein. SDS- PAGE and Western blot identification, and test its antioxidant activity. Results Recombinant plasmid pPIC9K-EgTPx was constructed and confirmed by restriction enzyme digestion and sequencing. The molecular mass unit of the expressed protein was about 22ku. The protein was recognized by mouse anti-EgTPx polyclonal antibody and had strong anti-oxidative activity. Conclusion EgTPx was successfully expressed in Pichia pastoris expression system, which has immunoreactivity and anti-oxidative activity.