金黄色葡萄球菌毒力因子PSM-α的4个基因串联融合表达及其对人中性粒细胞的生物学作用

摘要

Phenol-soluble modulin (PSM) is a newly identified secreted virulence factor of Staphylococcus aureus. To study the effect of the subunits of PSM-α, the genes of PSM-α1, PSM-α2, PSM-α3 and PSM-α4 were cloned and the recombinant plasmid pGEX-PSM-α was constructed for expression in E.coli. The highly expressed soluble protein were purified and digested with thrombin to remove the GST-tag. Compared with negative control, the expression of human neutrophils IL-8 was increased significantly after co-culture with the purified PSM-α detected by ELISA kit. The current study demonstrated that PSM-α was able to cause disease by stimulating human neutrophils to release inflammatory factor.%酚可溶性调控蛋白(PSM)是一种新发现的金黄色葡萄球菌(S.aureus)细胞外分泌蛋白,为研究其对人中性粒细胞的生物学作用,本研究通过设计linker将其中编码PSM-α蛋白的PSM-α1、PSM-α2、PSM-α3、PSM-α4基因串联,并克隆于pGEX-4T-1中.重组菌经IPTG诱导,重组蛋白以可溶形式高效表达.通过Glutathione Sepharose 4B亲和层析柱纯化并用凝血酶切除其GST标签,得到的蛋白作用于正常人的中性粒细胞,通过ELISA检测细胞因子IL-lβ及IL-8的含量.结果显示,纯化后蛋白刺激中性粒细胞后,IL-8含量显著升高.结果证明,串联表达后的PSM-α蛋白可以刺激中性粒细胞释放炎性因子,诱导炎症的发生,产生致病作用.