利用T7和PR双启动子的新型表达载体的构建及其应用

Construction and application of an expression vectors with combined T7 and PR promoters for flexible induced expression of cloned DNA in E.coli

目的：构建具有λＰＲ与Ｔ７双启动子的新型原核表达载体ｐＤＯＧ，以便使用同一载体研究不同诱导条件下，重组蛋白在大肠杆菌中表达、降解以及折叠的情况，从而选择最佳的诱导条件。方法：采用ｐＢＶ２２０与ｐＥＴ－３表达载体作为原始材料，将ｐＥＴ－３上包括Ｔ７启动子与Ｔ７ｇ－１０翻译起始序列的一段序列克隆到ｐＢＶ２２０载体λＰＲ启动子的下游，构建了双启动子表达载体ｐＤＯＧ。结果：ｐＤＯＧ载体含有λＰＲ与Ｔ７两个启动子，可以选择性地使用热诱导或在较低温度下使用化学诱导表达重组蛋白。该载体带有三个相位的多克隆位点，外源基因可以融合载体的少数几个氨基酸表达，也可以直接表达。该载体已成功地用于ＨＩＶ－１核心蛋白、人ＩＬ－６等外源基因的高效表达。

Objective: To construct novel E.coli expression vector that combines T7 and PR promoters for comparing degradation or refolding of the recombinant products under different induction conditions using the same vector. Methods: pBV220 And pET 3 are used as parent plasmids for the construction of the expression vector pDOG. This vector is generated by cloning the T7 g 10 leader sequence including T7 promoter and translation initiation region that is derived from pET 3 expression vector, downstream the λPR promoter of pBV220 vector. Results: pDOG Vector has PR and T7 promotes in tandem. Expression of foreign cDNA from the pDOG can be induced either by temperature induction, or by chemical induction at lower temperature. In addition, such vector contains multiple cloning sites where foreign cDNA can be inserted for expression as a fusion protein with 12 amino acids fused at its N terminus, and alternatively foreign gene can be cloned into NdeⅠ site for direct expression. The vector has been proved to be successful in over expression HIV 1 gag fragments and human IL 6. Conclusion: pDOG Vector has been demonstrated to be a valuable tool for optimizing expression of foreign gene in E.coli.