布鲁氏菌外膜蛋白BP26细胞毒性作用的研究

【目的】布鲁氏菌外膜蛋白BP26是布鲁氏菌的重要毒力因子，本研究采用bp26基因缺失株和BP26蛋白分别与胚胎滋养层细胞(HPT-8)作用，探讨布鲁氏菌bp26基因的生物学功能。【方法】采用Ni柱亲和层析法纯化BP26蛋白，overlap技术构建布鲁氏菌bp26基因缺失株，用BP26蛋白和bp26基因缺失株分别侵染HPT-8细胞，观察细胞形态变化，ELISA检测上清中的细胞因子。【结果】 从布鲁氏菌疫苗株M5-90克隆出bp26基因并在大肠杆菌E.coli BL21(DE3)中成功表达，获得纯化的BP26蛋白，经SDS-PAGE验证正确，Western-Blot鉴定具有免疫原性。将目的片段bp26基因的上下臂插入到自杀载体pGEM-7zf中，电转至布鲁氏菌疫苗株M5-90感受态细胞中，成功筛选出了具有遗传稳定性的bp26基因缺失株。用BP26蛋白与bp26基因缺失株分别侵染HPT-8层细胞，BP26蛋白使细胞变形且贴壁不牢，缺失株导致细胞大量脱落溶解，ELISA检测蛋白侵染HPT-8细胞诱导产生的细胞因子IL-6、TNF-α和LDH均高于PBS对照组，差异极显著（P＜0.01）, 而细胞因子IL-10的分泌下降。缺失株侵染HPT-8细胞诱导产生的细胞因子IL-6、TNF-α均高于M5-90对照组，LDH和IL-10均低于M5-90对照组，差异显著（P＜0.05）。【结论】成功获得了布鲁氏菌BP26蛋白和M5-90Δbp26缺失株，BP26蛋白可以引起HPT-8细胞的炎性反应，有细胞毒性作用，bp26基因缺失株对HPT-8细胞有损伤作用，bp26基因在布鲁氏菌侵染宿主细胞过程中起重要作用。

Study on Cytotoxicity of Outer Membrane Protein BP26 of Brucella

【Objective】Outer membrane protein BP26 of Brucella is an important Brucella virulence factor.In order to reveal the biological functions of Brucella bp26 gene,HPT-8 cells were infected by bp26 gene deletion strains and treated by BP26 protein respectively.【Method】The BP26 protein was purified by Ni affinity chromatography and bp26 gene deletion strains was constructed by overlap technology.After the infection of bp26 gene deletion strains and the treatment of BP26 protein,HPT-8 cells were observed by electron microscope to examine the cells morphology.ELISA was used to detect cytokines in the supernatant.【Results】bp26 gene was cloned from Brucella vaccine strain M5-90 and successfully expressed in E.coli BL21(DE3).Purified protein was proved correct by SDS-PAGE,and the immunogenicity of the obtained BP26 was confirmed by Western-blot.The upper and lower arms of bp26 gene were inserted into the suicide vector PGEM-7zf,transferred to Brucella vaccine strains M5-90 and the bp26 deletion strains with genetic stability were selected successfully.The treatment of BP26 protein changed the cells morphology and significantly decreased the adhesion ability to the wall.Compared with the PBS group,the release level of IL-6,TNF-α and LDH significantly increased(P<0.01),but the release level of IL-10 significantly decreased(P<0.05).After the infection of bp26 gene deletion strains,the release level of IL-6,TNF-α were higher than M5-90 group.The release level of LDH and IL-10 were significantly lower than M5-90 group(P<0.05).【Conclusion】BP26 protein and Brucella M5-90Δbp26 deletion strains were successfully obtained.BP26 protein could lead to inflammatory response and had some toxic effects on the HPT-8 cells.The bp26 gene plays an important role in Brucella infection of HPT-8 cells.