人铜锌超氧化物歧化酶真核表达载体的构建及在酿酒酵母中的分泌表达

Constraction of eukaryotic expression vector of human Cu/Zn-SOD and secretive expression in S.cerevisiae

目的:探讨酿酒酵母中人铜锌超氧化物歧化酶(hCu/Zn-SOD)的分泌表达。方法:采用RT-PCR技术从人胃总RNA中分离扩增hCu/Zn-SOD的cDNA,与酿酒酵母交配因子(MF)α信号肽编码序列融合,将融合基因插入大肠杆菌-酵母细胞穿梭型质粒pYES2.0,构建真核分泌表达质粒pYES-MS,将其转化入酿酒酵母的缺陷型菌株INVSc1中,用半乳糖进行诱导表达,采用SDS-PAGE、蛋白质免疫印迹分析及电泳谱活性染色测定表达产物分泌情况及其活性。结果:该工程菌可高效表达一相对分子质量约为21000的蛋白并与兔抗人Cu/Zn-SOD多抗有特异的免疫反应,同时在培养基上清中检测到此表达蛋白的分泌,具有特异性SOD酶活性。结论:作者成功构建了pY-ES-MS/INV菌株,该菌株大量表达有天然酶活性的hCu/Zn-SOD,并有部分hCu/Zn-SOD分泌到酵母细胞外,为开发hCu/Zn-SOD的基因工程产品奠定了良好的基础。

Aim ： To investigate the secretive expression of human Cu/Zn-SOD in saccharomyces cerevisiae （S. cerevisiae）. Methods：The cDNA encoding human Cu/Zn-SOD was amplified by RT-PCR using the total mRNA of human stomach as the template. The cloned human Cu/Zn-SOD eDNA with the yeast mating factor（MF） α signal sequence was constructed into an E. coli/ S. cerevisiae shuttle vector pYES2.0 resulting in recombinant expression plasmid pYES-MS which was then thansformed into S, cerevisiae INVScl. The transformant was cultured in YPD culture media and induced with 2% galactose. Results： The engineering S. cerevisiae highly expressed a protein with relative molecular weight of 21 000. SDS-PAGE and Western-blot analysis revealed that partial SOD was secreted into the culture broth, while most of the protein was found in the yeast cells. Conclusion ： The engineering S. eerevisiae expressing human Cu/Zn-SOD is established, which lays a good basis of large scale purification and further practical research of hCu/Zn-SOD.