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  

Molecular regulatory mechanism of two-component regulatory system SaeRS on important virulence genes in Staphylococcus aureus

作　　者：许园园[1] 丁宇[2] 刘云灵 李丹[2] 王良兴[3] 余方友[2] 

机构地区：[1]宁波市医疗中心李惠利医院呼吸内科,浙江宁波315041 [2]温州医科大学附属第一医院检验科,浙江温州325015 [3]温州医科大学附属第一医院呼吸内科,浙江温州325015

出　　处：《温州医科大学学报》2015年第9期625-630,共6页Journal of Wenzhou Medical University

基　　金：国家自然科学基金资助项目(81271906H2002)

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。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 pET28 a,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 NiNTA 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 levels of luk-PV,hla,coa,fnbB mRNA of SA75 AsaeRS mutant strain decreased to 19.7%,0.3%,31.6%and 3.5%respectively whereas the levels of psmβand 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.

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 

分 类 号：R378.1[医药卫生—病原生物学]