一种带增强子的原核高效表达载体的构建及初步应用

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50%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。

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

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 tobacco mosaic virus (TMV), omega sequence, was ligated in front of a T7 promoter in the regulatory sequence; (2) the multi-cloning sites include eight restriction enzyme sites. It can facilitate fusion or nonfusion 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 yield of target gene from plasmid pTO-T7 compared with that from pT-T7 without omega sequence suggested that omega sequence in pTO-T7 can improve the expression of target gene significantly.