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A Stable Vector for High-Level Expression and Secretion of Human Interferon αA in Yeast 总被引:1,自引:0,他引:1
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. 相似文献
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以硒蛋白K(SelK)突变体为"诱饵", 采用酵母双杂交系统对人肝cDNA文库进行筛选, 得到一个与SelK相互作用的蛋白──环腺苷酸应答元件结合蛋白3(CREB3). 将SelK与CREB3共同转染酵母细胞, 验证了SelK与CREB3的相互作用; 并采用受体漂白、敏化发射和荧光寿命3种荧光共振能量转移方法进一步验证了二者间的相互作用, 发现其不受SelK中硒代半胱氨酸(Sec)的影响. 推测SelK可能通过其Sec之前的区域与CREB3发生作用, 参与CREB3介导的内质网相关降解过程, 影响相关癌症的转移和发展. 相似文献
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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 总被引:8,自引:0,他引:8
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。 相似文献
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