细粒棘球绦虫硫氧还蛋白过氧化物酶基因在毕赤酵母中的分泌表达及生物学功能鉴定

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。

Characterization of Echinococcus granulosus thioredoxin peroxidase expressed in Pichia pastoris

Objectives To express Echinococcus granulosus thioredoxin peroxidase （EgTPx） in a secretory form in Pichia pastoris and to determine its activity. Methods Total RNA was extracted from protoscoleces isolated from an E. granulosus cyst from a sheep, cDNA was synthesized from the RNA and the EgTPx gene was amplified with PCR using cDNA as a template. The amplified DNA fragment was subcloned into an expression vector pPIC9K designated pPICgK EgTPx. The recombinant plasmid was linearized and transformed into GS115 strain Pichia pastoris yeast using electroporation. Multiple copies of the transformants were screened with G418 and His＋ clones were screened on MD agar plates. EgTPx insert DNA was identified using PCR analysis and the open reading frame was confirmed by sequencing. Under the control of the promoter AOX1, the gene was induced with methanol to secret EgTPx into culture medium. Results Both restriction analysis and sequencing indicated that the recombinant plasmid pPIC9K-EgTPx was constructed correctly. The expressed EgTPx protein had a relative molecular mass of about 22 ku. The protein hound specifically to mouse anti-EgTPx polyclonal antibody and it had a high level of antioxidant activity. Conclusion To the extent known, this is the first time that the EgTPx gene was successfully expressed in P. pastoris in a secretory form.