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目的探讨赖型钩端螺旋体Loa22蛋白对血管内皮的毒性作用和功能影响。方法用赖型钩端螺旋体Loa22成熟肽原核重组质粒进行诱导表达带有GST标签的Loa22融合蛋白,经亲和层析柱进行纯化并获取目标蛋白,用SDS-PAGE和Western blot对目标蛋白进行检测和证实。用获得的GST-Loa22融合蛋白刺激处理培养的人脐静脉血管内皮细胞(HUVEC)来说明钩端螺旋体外膜蛋白Loa22对血管内皮细胞的毒性作用以及对其通透性的影响。结果成功表达Loa22成熟肽原核重组质粒,通过鉴定并纯化得到Loa22融合蛋白,该蛋白随着浓度的升高能使培养的血管内皮细胞CCK-8吸光度值降低,细胞凋亡率增加,HUVEC单层通过率升高。结论Loa22融合蛋白对血管内皮细胞有明显的毒性作用,还使得HUVEC通透性增加。
Objective To investigate the toxic effect and function of LoA22 leptospira on vascular endothelial cells. Methods Loa22 fusion protein with GST-tag was induced by the LoA22 mature peptide of Leptospira interrogans. The recombinant protein was purified by affinity chromatography and the target protein was obtained. The target protein was detected by SDS-PAGE and Western blot And confirmed. The cultured human umbilical vein endothelial cells (HUVECs) were stimulated with the obtained GST-Loa22 fusion protein to explain the toxic effect of the leptospiral outer membrane protein Loa22 on the vascular endothelial cells and its permeability. Results Loa22 mature peptide prokaryotic recombinant plasmid was successfully expressed. After Loa22 fusion protein was identified and purified, the CCK-8 absorbance value of cultured endothelial cells decreased with the increase of concentration, and the apoptosis rate of HUVEC monolayer increased Rate increased. Conclusion The Loa22 fusion protein has obvious toxic effect on vascular endothelial cells and also increases the permeability of HUVEC.