本文關(guān)鍵詞： 金黃色葡萄球菌 雙組份調(diào)節(jié)系統(tǒng) saeRS 反應(yīng)調(diào)節(jié)蛋白SaeR　出處：《溫州醫(yī)科大學(xué)學(xué)報(bào)》2015年09期 　論文類(lèi)型：期刊論文

【摘要】：目的:初步研究金黃色葡萄球菌雙組份調(diào)節(jié)系統(tǒng)Sae RS對(duì)重要毒力基因的分子調(diào)控機(jī)制。方法:PCR擴(kuò)增雙組份調(diào)節(jié)系統(tǒng)Sae RS的反應(yīng)調(diào)節(jié)蛋白Sae R基因,構(gòu)建重組表達(dá)質(zhì)粒pET28a-sae R,用限制性酶切、PCR和基因測(cè)序方法進(jìn)行鑒定。用IPTG誘導(dǎo)表達(dá)重組蛋白His Sae R,用鎳離子螯合親和層析法純化,用Western blot法鑒定。用實(shí)時(shí)熒光定量RT-PCR方法檢測(cè)金黃色葡萄球菌SA75及SA75Δsae RS突變株luk-PV、hla、coa、fnb B、sak、psmβ基因轉(zhuǎn)錄水平。凝膠遷移阻滯實(shí)驗(yàn)驗(yàn)證純化蛋白His Sae R對(duì)這些毒力基因啟動(dòng)區(qū)序列的結(jié)合能力。結(jié)果:重組表達(dá)質(zhì)粒p ET28a-sae R構(gòu)建成功;在0.4 mmol/L IPTG,25℃培養(yǎng)12 h的條件下,重組蛋白His Sae R在大腸桿菌中以可溶形式高效表達(dá);與SA75野生株相比,Dsae RS突變株luk-PV、hla、coa、fnb B基因轉(zhuǎn)錄水平均明顯下降,分別為野生株的19.7%、0.3%、31.6%、3.5%;psmβ基因和sak基因轉(zhuǎn)錄水平無(wú)變化。反應(yīng)調(diào)節(jié)蛋白Sae R能有效結(jié)合luk-PV、hla、coa、fnb B及其自身P1啟動(dòng)區(qū)序列。結(jié)論:金黃色葡萄球菌雙組份調(diào)節(jié)系統(tǒng)Sae RS對(duì)luk-PV、hla、coa、fnb B基因表達(dá)具有正調(diào)控作用,而這種作用可能是通過(guò)反應(yīng)調(diào)節(jié)蛋白Sae R與這些基因啟動(dòng)區(qū)序列直接結(jié)合而實(shí)現(xiàn)。
[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，

本文編號(hào)：1495597