大豆24 kDa油体蛋白基因在大肠杆菌中的表达.pdfVIP

中国农业科学 2005,38(2):234-240 Scientia Agricultura Sinica 大豆24 kDa油体蛋白基因在大肠杆菌中的表达 徐妙云，刘德虎，李刚强 （中国农业科学院生物技术研究所，北京 100081 ） 摘要：分别构建了含全长大豆 24 kDa 油体蛋白基因、与大肠杆菌热敏毒素 B 亚基和定居因子 CS6 B 亚基基 因以串联方式融合的全长大豆 24 kDa 油体蛋白基因、缺失 N 端 74 个氨基酸编码序列的油体蛋白基因及缺失 C 端 152 个氨基酸编码序列油体蛋白基因等 4 个大肠杆菌表达载体。除 N 端缺失的油体蛋白基因外，其它 3 个载体的 IPTG 诱导表达产物对 E.coli 生长表现出明显的抑制作用，菌液浓度与对照菌株相比，至少降低了 3 倍；而 N 端 缺失了 222 个编码核苷酸的油体蛋白基因则对宿主细菌细胞的生长繁殖不再表现有任何毒性。进一步通过 SDS和Western印迹反应检测各表达载体的IPTG诱导表达产物时发现，仅有N端缺失的24 kDa 油体蛋白基 因片段在宿主细胞BL21(DE3)和Rosseta(DE3)中有重组油体蛋白积累。 关键词：大豆；油体蛋白；表达；大肠杆菌 Expression of Soybean 24 kDa Oleosin Gene in Escherichia coli XU Miao-yun, LIU De-hu, LI Gang-qiang (Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081) Abstract : Four bacterial expression vectors were constructed, which carry the full-length, the N-end truncated, the C-end truncated soybean 24 kDa oleosin gene as well as the fused fragment with the genes encoding the heat-labile toxin B subunit and CS6 B subunit from E.coli . The growth of host cells was depressed when the full-length 24 kDa oleosin gene was expressed in E. coli either as the native fragment or as a tandem unit fused with bacterial genes, with a reduction to about one-third of the growth rate of control culture. Deletion of the DNA sequence encoding 152 amino acids near the C terminus of the 24 kDa oleosin could not overcome this toxicity. However, the toxicity can be overcomed by deletion of the sequence encoding the 74 amino acids of the N terminal region of the protein. When products induced by IPTG were detected by SDSand Western blotting, only the 24 kDa oleosin gene fragment tru