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Hsh载体对外源基因在E. coli中高效表达的机制探讨
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长江大学博士启动基金项目(No. 801100010112)


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

    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。

    Abstract:

    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.

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吴华伟,邵蔚蓝. Hsh载体对外源基因在E. coli中高效表达的机制探讨[J]. 微生物学通报, 2011, 38(9): 1355-1361

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