大肠杆菌yijP侵袭蛋白诱导人脑微血管内皮细胞凋亡

为研究大肠杆菌的脑微血管内皮细胞侵袭基因yijP的功能，将yijP基因（1．04kb）克隆到pQE30表达载体，构建表达产物为N末端带有6个组氨酸（His）序列的yijP汇合蛋白，以M15（pREP4）为受体菌，大量表达（His）6-yijP汇合蛋白，利用Ni—NTA亲和层析纯化汇合蛋白，将经透析法复性的一定浓度的（His）6-yijP蛋白加入到体外培养的人脑微血管内皮细胞中，结果显示yijP蛋白对人脑微血管内皮细胞有较强的细胞毒作用：在相差显微镜下可观察到细胞皱缩、胞膜呈泡状膨出，随着时间延长细胞逐渐脱落；荧光显微镜下可见细胞核呈现为致密团块状或圆形浓染颗粒状，呈凋亡样改变：DNA琼脂糖凝胶电泳可见DNA阶梯状条带；流式细胞仪显示在正常二倍体峰之前出现一个亚二倍体峰；Western印迹可检测到caspase-3的活性片段。这些现象均出现在yijP蛋白作用于人脑微血管内皮细胞的16h之后，提示在大肠杆菌侵袭人脑微血管内皮细胞过程中，yijP蛋白可能起到诱导脑微血管内皮细胞迟发性凋亡的毒素作用。

Escherichia coli Invasion Protein yijP Induces Apoptosis of Human Brain Microvascular Endothelial Cells

In order to further characterize the role of invasion locus yijP in the pathogenesis of E. coli meningitis, the yijP gene (about 1.04 kb) was subcloned into pQE30, and expressed in E.coli M15(pREP4) as His- tagged confluence protein induced by IPTG. The successfully harvested confluence protein by Ni-NTA agarose affinity chromatography was refolded by dialysis. Phase contrast microscopy was used to observed the morpho- logical changes of human brain microvascular endothelial cells (HBMEC), and Hoechst 33258 staining, DNA agar- ose gel electrophoresis, FACScan flow cytometer and Western blotting were employed to investigate the type of the cell death. The results showed that the confluence protein yijP was toxic to HBMEC. Besides the cell shrinkage and nuclear condensation, DNA fragmentation, a sub-diploid peak and activity cleavage of caspase-3 were detected, which were the characteristics of the cell apoptosis. All these phenomena occurred after 16-hour incubation with the yijP. Our results indicated that yijP protein may act as a kind of toxin to induce delayed apoptosis of HBMEC.