Original Article
Prokaryotic expression, reactogenicity, and function of Brucella secretary protein BspE
Shuanghong Yin, Junbo Zhang, Hong Zhang, Xiulan Shi, Jihai Yi, Huan Zhang, Chuangfu Chen, Zhiqiang Li
Published 2018-09-20
Cite as Chin J Endemiol, 2018, 37(9): 689-694. DOI: 10.3760/cma.j.issn.2095-4255.2018.09.001
Abstract
ObjectiveTo investigate the prokaryotic expression and immunoreactivity of BspE, a type Ⅳ secretion protein of Brucella, and the effect of recombinant protein BspE on cytokines.
MethodsAccording 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.
ResultsThe 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).
ConclusionsBspE 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.
Key words:
Brucella; BspE gene; Prokaryotic expression; Inflammatory factors
Contributor Information
Shuanghong Yin
Department of Nursing, College of One Health, Tongren University of Guizhou, Tongren 554300, China
Junbo Zhang
Department of Animal Microbiology, College of Agroforestry Engineering and Planning, Cultural and Technological Industry Innovation Research Center, Tongren University of Guizhou, Tongren 554300, China
Hong Zhang
Department of Nursing, College of One Health, Tongren University of Guizhou, Tongren 554300, China
Xiulan Shi
Department of Nursing, College of One Health, Tongren University of Guizhou, Tongren 554300, China
Jihai Yi
Department of Preventive Veterinary Medicine, College of Animal Science and Technology, Shihezi University of Xinjiang, Shihezi 832002, China
Huan Zhang
Department of Preventive Veterinary Medicine, College of Animal Science and Technology, Shihezi University of Xinjiang, Shihezi 832002, China
Chuangfu Chen
Department of Preventive Veterinary Medicine, College of Animal Science and Technology, Shihezi University of Xinjiang, Shihezi 832002, China
Zhiqiang Li
Department of Microbiology, College of Biotechnology and Food, Shangqiu Normal University of Henan, Shangqiu 476000, China
