一种高效、稳定的分泌型原核表达载体的构建及应用

以本室构建的原核表达载体pTO-T7为基础载体，PCR合成ompT引导序列，插入该载体多克隆位点上游，构建了分泌型原核表达载体pTO—OT。将2个外源基因克隆至pTO—OT，2个重组质粒在大肠杆菌中均得以高效表达，表达量为25％～30％。Western印迹分析证实了重组蛋白在大肠杆菌中表达后可被信号肽酶有效识别，切割后的重组蛋白具有良好的免疫学活性。对重组表达菌株的连续传代实验证实了该表达载体具有良好的遗传稳定性，显示了该原核表达载体在基因工程中的应用价值。

Construction and application of an efficient, stable secretion prokaryotic expression vector

A secretion prokaryotic expression vector, pTO-OT, was constructed based on the high-level expression vector pTO-T7 by inserting ompT leader into the MCS of pTO-T7. Two foreign genes were cloned into pTO-OT and could be expressed efficiently in E.coli. The ratio of every recorabinant protein to total bacteria proteins varied from 25% to 30%. Western blot analysis on the two recombinant proteins suggested that they could be recognized and cut by signal peptidase in E.coli, and the mature proteins were immuoactive. The consecutive culture of recombinant engineering strains showed pTO-OT has excellent genetic stability, which suggested pTO-OT is a practical vector in the future genetic engineering.