A Stable Vector for High-Level Expression and Secretion of Human Interferon αA in Yeast

Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10~(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.     

酵母高稳定载体的构建及人-αA干扰素在酵母中的高表达和分泌

本文利用酿酒酵母天然2μm质粒的SnaBI位点与pEMBL Yi27质粒的SmaI位点经平末端连接,构建成酿酒酵母高稳定质粒载体pHC11,经测定表明,其酵母转化子在非选择培养条件下连续生长50世代后,仍有82％的细胞保留该质粒,此结果证实了2μm质粒中非功能区的存在,将人-αA干扰素基因表达分泌单元插入pHC11,构建成重组质粒转化酵母后,在完全培养基中发酵培养,经分析测定,培养上清液中表达产物占总蛋白量的36.8％,干扰素效价达2.6×10~(10)u/L,表明利用高稳定载体pHC11使人-αA干扰素在酵母中得到了高表达和分泌。