摘要目的 观察布鲁菌Ⅳ型分泌系统效应蛋白BspE的原核表达、免疫反应原性,以及重组蛋白BspE对细胞因子的影响.方法 根据GenBank中公布的羊种布鲁菌M5-90的BspE基因,合成基因片段,将其连接到PUC57载体测序,将测序正确的基因序列克隆到原核表达载体pET-28α上,转化入大肠埃希菌DE3感受态细胞中诱导表达,Ni-NTA亲和柱纯化目的蛋白,蛋白质免疫印迹(Western blot)法分析其反应原性.25 g/LBspE重组蛋白作用小鼠巨噬细胞系RAW264.7细胞,对照组采用相同含量的牛血清白蛋白(BSA)替代BspE,作用12、24、48 h,酶联免疫吸附试验检测白细胞介素(IL)-1β水平.结果 成功获得pET-28α-BspE重组表达质粒;Western blot结果显示,出现相对分子质量约为30.1×103的条带,纯化后条带单一,BspE重组蛋白具有较好的反应原性.与BSA组比较,BspE蛋白可显著提高IL-1β (ng/L)的水平(12h:4327±213比30.24±1.66、24 h:57.78±3.44比41.22±1.22、48 h:72.52±3.04比46.77±2.75,t=8.38、7.86、10.89,P均＜0.05).结论 BspE原核表达后具有较好的免疫反应原性,并可提高小鼠巨噬细胞IL-1β的表达水平,为效应蛋白在布鲁菌致病机制中作用的研究提供科学依据.

abstractsObjective 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.