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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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

Received: 31 December 2014

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	XU Yuanyuan1
	DING Yu2
	LIU Yunling3
	LI Dan2
	WANG Liangxing3
	YU Fangyou2.

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https://xb.wmu.edu.cn/EN/ OR https://xb.wmu.edu.cn/EN/Y2015/V45/I9/625

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