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

发布时间：2018-02-08 13:52

  本文关键词： 金黄色葡萄球菌 双组份调节系统 saeRS 反应调节蛋白SaeR　出处：《温州医科大学学报》2015年09期 　论文类型：期刊论文

【摘要】：目的:初步研究金黄色葡萄球菌双组份调节系统Sae RS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Sae RS的反应调节蛋白Sae R基因,构建重组表达质粒pET28a-sae R,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 Sae R,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SA75Δsae RS突变株luk-PV、hla、coa、fnb B、sak、psmβ基因转录水平。凝胶迁移阻滞实验验证纯化蛋白His Sae R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p ET28a-sae R构建成功;在0.4 mmol/L IPTG,25℃培养12 h的条件下,重组蛋白His Sae R在大肠杆菌中以可溶形式高效表达;与SA75野生株相比,Dsae RS突变株luk-PV、hla、coa、fnb B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Sae R能有效结合luk-PV、hla、coa、fnb B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Sae RS对luk-PV、hla、coa、fnb B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Sae R与这些基因启动区序列直接结合而实现。
[Abstract]:Objective: to study the molecular regulation mechanism of staphylococcus aureus bicomponent regulatory system Sae RS on important virulence genes. Methods: the reaction-regulating protein Sae R gene of two-component regulatory system Sae RS was amplified by PCR. The recombinant expression plasmid pET28a-sae R was constructed and identified by restriction enzyme digestion and gene sequencing. The recombinant protein His Sae Rwas induced by IPTG and purified by nickel ion chelating affinity chromatography. The transcriptional level of staphylococcus aureus SA75 and SA75 螖 sae RS mutant luk-PVhlaafnb Brankakakanfm 尾 was determined by Western blot method. The sequence of promoter region of purified His Sae R to these virulence genes was confirmed by gel migration block assay. Results: the recombinant expression plasmid p ET28a-sae R was successfully constructed. The recombinant protein His Sae R was highly expressed in Escherichia coli at 25 鈩，

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