赖型钩端螺旋体OmpA外膜蛋白Loa22对血管内皮细胞的毒性及对通透性的影响  

The Cytotoxic Effect and the Effect of Permeability with OmpA Like Protein Loa22 from Leptospira interrogans Serovar Lai on HUVEC

作　　者：王一舟[1] 余德立[1] 夏志杨[1] 鲍朗[1] 

机构地区：[1]四川大学华西基础医学与法医学院感染免疫实验室,成都610041

出　　处：《四川大学学报（医学版）》2015年第2期169-172,共4页Journal of Sichuan University(Medical Sciences)

摘　　要：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 study the toxic effect and the change of permeability on human umbilical vein endothelia（HUVE）of the Loa22 protein from virulent serovar Lai.Leptaspira interrogans by expressing its protein.Methods In this study,the pGEX-Loa22 peptide prokaryotic recombinant plasmid of Leptospira interrogans serovar Lai preserved in our laboratory was used to express Loa22 fusion protein with GST lable.Then the target fusion protein was obtained by using affinity chromatography with the GST-Trap FF Column.The purified Loa22 fusion protein was detected by SDS-PAGE and confirmed by Western blot assay using the mouse antiGST tag monoclonal anti-body.pGEX-Loa22 protein was administered to culture with human umbilical vein endothelial cells（HUVEC）to elucidate the cytotoxic role and the change of permeability of leptospiral outer membrane proteins.Results The recombiant plasmid with Loa22 mature peptide was expressed successfully and the protein was purfied.Significant higher level of apoptosis ratio,lower CCK-8aborntion,and increasing permeability on HUVEC were observed after treated the HUVEC with the expressed fusion protein.Conclusion The purified Loa22 fusion protein have obvious toxic effects on vascular endothelial cells,and also it can increase permeability of HUVEC.

关 键 词：赖型钩端螺旋体 Loa22 人脐静脉血管内皮细胞 细胞毒性 凋亡 

分 类 号：R514.4[医药卫生—内科学]