pHsh载体对外源基因在E.coli中高效表达的机制探讨

Mechanism of high level expression of foreign gene caused by pHsh vector in Escherichia coli

pHsh是根据大肠杆菌的热休克反应构建而成的新型表达载体,受σ32调控。正常E.coli细胞的整个热休克反应持续时间约12 min,而在携带有外源基因的高拷贝pHsh的E.coli细胞中,外源基因却能持续高效表达4 10 h。为探求外源基因高效表达的机制,以一个编码木聚糖酶的外源基因为代表,首先研究了质粒拷贝数对木聚糖酶表达的影响,接着通过Western-blot检测了携带质粒pHsh-xynIII和对照组携带pLac-xynIII的E.coli细胞在非诱导条件下(30°C)和诱导条件下(30°C→42°C)胞内σ32的差异,最后测定了不同温度下(30°C、37°C、42°C、30°C→42°C)携带质粒(pHsh-xynIII)的E.coli细胞内稳定状态下热休克的水平(以木聚糖酶活性表征)。研究结果表明外源基因在pHsh中的高效表达是与3个方面密切相关的:pHsh质粒的高拷贝数增加了外源基因的剂量;pHsh的存在使E.coli细胞内σ32的水平较正常E.coli细胞显著增加了,并最终增强了E.coli的热休克反应;诱导状态下带有pHsh重组质粒的E.coli细胞内稳定状态下的热休克水平明显高于其它温度的水平。

pHsh is a novel high level expression vector of Escherichia coli,in which the regulatory promoters are recognized by the 32-kD sigma factor（σ32）.In normal E.coli cells,the total time length of heat-shock response is about 12 min,however,in E.coli cells carrying recombinant high-copy pHsh vectors,the heat-shock response can sustain 4-10 h.In order to understand the mechanism of hign level expression of foreign gene in E.coli carrying pHsh vector,we employed xynIII gene encoding a xylanase as the representative of foreign genes.Firstly,the effect of copy-number of pHsh on the expression level of xynIII gene was tested,then the difference of the concentration of σ32 between in the E.coli cells harboring pHsh-xynIII and in the E.coli cells harboring pLac-xynIII was assayed by using western-blot under either inducing（30 °C→42 °C） or non-inducing conditions（30 °C）.Finally,under different temperatures,the heat-shock level at steady state in recombinant E.coli cells harboring pHsh-xynIII was evaluated by the xylanase activity.the results showed that the high expression level of foreign genes in pHsh should attribute to the following three aspects： high-copy-number of pHsh enhanced the foreign gene dosage accessible for expression and leaded to a high productivity;Owing to the present of the pHsh,σ32 level in E.coli cells was significantly higher than that in E.coli cells without pHsh,thus the heat-shock level was significantly enhanced in E.coli cells harboring pHsh;The considerable heat-shock level at steady state in recombinant E.coli cells harboring pHsh was helpful for high level expression of foreign genes.