Prokaryotic expression, reactogenicity, and function of Brucella secretary protein BspE
10.3760/cma.j.issn.2095-4255.2018.09.001
- VernacularTitle:布鲁菌分泌蛋白BspE原核表达、反应原性及功能初步研究
- Author:
Shuanghong YIN
1
;
Junbo ZHANG
;
Hong ZHANG
;
Xiulan SHI
;
Jihai YI
;
Huan ZHANG
;
Chuangfu CHEN
;
Zhiqiang LI
Author Information
1. 贵州省铜仁学院大健康学院护理学教研室
- Keywords:
Brucella;
BspE gene;
Prokaryotic expression;
Inflammatory factors
- From:
Chinese Journal of Endemiology
2018;37(9):689-694
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the prokaryotic expression and immunoreactivity of BspE,a type Ⅳ secretion protein of Brucella,and the effect of recombinant protein BspE on cytokines.Methods According to the BspE gene of Brucella M5-90 published in GenBank,the gene fragments were synthesized by a company and then ligated into PUC57 vector for sequencing.The sequenced gene was cloned into a prokaryotic expression vector pET-28α and transformed.Induced expression was performed in E.coli DE3 competent cells.The obtained target protein was purified by a Ni-NTA affinity column,and its reactogenicity was analyzed by Western blotting.Mouse RAW264.7 cells were treated with 25 g/L BspE recombinant protein for 12,24,48 h,and the control group was treated with the same amount of BSA instead of BspE,and enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin (IL)-1β level.Results The recombinant expresed plasmid of pET-28α-BspE was successfully obtained.The results of Western blotting showed a single band with a relative molecular mass of about 30.1 × 103,and the recombinant protein BspE had good reactogenicity,and IL-1β levels (ng/L)were significantly elevated by the recombinant protein BspE (12 h:43.27 ± 2.13 vs 30.24 ± 1.66,24 h:57.78 ± 3.44 vs 41.22 ± 1.22,48 h:72.52 ± 3.04 vs 46.77 ± 2.75,t =8.38,7.86,10.89,P ＜ 0.05).Conclusions BspE recombinant protein has better immunoreactivity and can increase the expression level of IL-1β in mouse macrophages.This study provides a scientific basis for the role of effector proteins in the pathogenesis of Brucella.
