金黄色葡萄球菌双组份调节系统SaeRS对重要毒力基因的分子调控机制

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摘要目的：初步研究金黄色葡萄球菌双组份调节系统SaeRS对重要毒力基因的分子调控机制。方法：PCR扩增双组份调节系统SaeRS的反应调节蛋白SaeR基因，构建重组表达质粒pET28a-saeR，用限制性酶切、PCR和基因测序方法进行鉴定。用IPTG诱导表达重组蛋白His SaeR，用镍离子螯合亲和层析法纯化，用Western blot法鉴定。用实时荧光定量RT-PCR方法检测金黄色葡萄球菌SA75及SA75ΔsaeRS突变株luk-PV、hla、coa、fnbB、sak、psmβ基因转录水平。凝胶迁移阻滞实验验证纯化蛋白His SaeR对这些毒力基因启动区序列的结合能力。结果：重组表达质粒pET28a-saeR构建成功；在0.4 mmol/L IPTG，25 ℃培养12 h的条件下，重组蛋白His SaeR在大肠杆菌中以可溶形式高效表达；与SA75野生株相比，∆saeRS突变株luk-PV、hla、coa、fnbB基因转录水平均明显下降，分别为野生株的19.7%、0.3%、31.6%、3.5%；psmβ基因和sak基因转录水平无变化。反应调节蛋白SaeR能有效结合luk-PV、hla、coa、fnbB及其自身P1启动区序列。结论：金黄色葡萄球菌双组份调节系统SaeRS对luk-PV、hla、coa、fnbB基因表达具有正调控作用，而这种作用可能是通过反应调节蛋白SaeR与这些基因启动区序列直接结合而实现。

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	许园园1
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	王良兴3
	余方友2

关键词 ：金黄色葡萄球菌, 双组份调节系统, saeRS, 反应调节蛋白SaeR

Abstract：Objective: To investigate the molecular regulatory mechanism of two-component regulatory system SaeRS on important virulence genes in Staphylococcus aureus. Methods: The 687-bp saeR gene was PCR amplified and cloned into pET28a, resulting in the plasmid pET28a-saeR, then tested by restriction enzyme analysis, PCR and gene sequencing. The recombinant protein His SaeR was induced by IPTG, purified with Ni-NTA agarose and verified by Western blot. The expression levels of luk-PV, hla, coa, fnbB, sak, psmβ genes were detected by real-time PCR. The binding abilities between SaeR and the promoter regions of virulence genes were tested by electrophoretic mobility shift assay. Results: Restriction enzyme digestion, PCR amplification and gene sequencing showed that the recombinant plasmid pET28a-saeR was successfully constructed. The recombinant protein His SaeR was efficiently expressed in soluble in E.coli BL21 (DE3) induced by 0.4 mmol/L IPTG at 25 ℃ for 12 hours. Compared to wild type strain SA75, the 1evels of luk-PV, hla, coa, fnbB mRNA of SA75ΔsaeRS mutant strain decreased to 19.7%, 0.3%, 31.6% and 3.5% respectively whereas the 1evels of psmβ and sak mRNA had no difference between wild type and mutant strain. The electrophoretic mobility shift assay showed that phosphorylated purified His SaeR could bind to the promoter regions of luk-PV, hla, coa, fnbB and P1 promoter. Conclusion: Two-component regulatory system SaeRS directly up-regulates the luk-PV, hla, coa, fnbB genes which is realized via response regulator protein SaeR combined with their promoter regions.

Key words： Staphylococcus aureus two-component regulatory system saeRS response regulator protein SaeR

收稿日期: 2014-12-31

基金资助:国家自然科学基金资助项目（81271906H2002）。

通讯作者: 余方友，副主任医师，副教授，Email：wzjxyfy@163.com。

作者简介: 许园园（1987-），女，浙江宁波人，住院医师，硕士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2015/V45/I9/625

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