布鲁氏菌外膜蛋白BP26细胞毒性作用的研究

doi:10.3864/j.issn.0578-1752.2012.16.021

Abstract

Abstract: 【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.

Key words: Brucellosis, BP26 protein, bp26 gene deletion strains, cytokine

CHEN Rui-Hua, ZHANG Hui, TANG Li-Yan, MENG Ru, ZHANG Yu, WANG Zhen, LI Zhi-Qiang, ZHANG Jun-Bo, CHEN Chuang-Fu. Study on Cytotoxicity of Outer Membrane Protein BP26 of Brucella[J].Scientia Agricultura Sinica, 2012, 45(16): 3406-3413.

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