一种带增强子的原核高效表达载体的构建及初步应用

Construction and Application of an Escherichia coli High Effective Expression Vector with an Enhancer

构建的 pTO T7大肠杆菌高效融合表达载体 ,调控序列中有一个Ω序列和一个T7启动子串联 ;多克隆位点 (MCS)包括 8个常用酶切位点 ;可进行融合表达或者非融合表达 ,根据不同的需要加以选择 ;融合蛋白N端为T7g10的 12个起始氨基酸 ,C端为His标签 ;含kan抗性基因作为选择标记。将增强型绿色荧光蛋白 (EGFP)基因克隆至 pTO T7载体 ,在E .coli中的表达结果表明 ,融合EGFP达到菌体总蛋白量的 5 0 %以上 ,90 %以上的融合蛋白以可溶性形式存在 ,融合后的EGFP仍保持原有的荧光性质。与同时构建的不含Ω序列的 pT T7载体的表达产量的比较研究结果表明 ,Ω序列在 pTO T7载体中能显著提高表达效率。

In this study,we constructed a high effective fusion expression vector in E.coli . This vector,pTO T7,was characterized as: (1) an enhancer from tobacoo mosaic virus (TMV),Ω sequence,was ligated in front of a T7 promoter in the regulatory sequence; (2) the multicloning sites include eight restriction enzyme sites. It can facilitate fusion or non fusion expression; (3) the N terminal of a fusion protein starts with the first 12 amino acids of T7 gene 10,and the C terminal is the hexahistidine tag; (4) kanmycin resistance gene was used as a selective marker. EGFP gene was inserted into pTO T7 vector as a reporter gene.Expression data showed that fused EGFP accounted to more than 50% of the total E.coli protein,and more than 90% of which was soluble. The fluorescence characters of fused EGFP were also studied. The expression yeild of target gene from plasmid pTO T7 compared with that from pT T7 without Ω sequence suggestted that Ω sequence in pTO T7 can improve the expression of target gene significantly.