布鲁氏菌核糖体L7/L12蛋白的原核表达及对鼠源树突状细胞的作用

为了研究和探讨布鲁氏菌核糖体L7/L12蛋白对鼠源树突状细胞(BM-DCs)分化和成熟的影响,用布鲁氏菌S2疫苗株为模板扩增L7/L12基因,构建重组质粒pET30a-L7/L12,用大肠埃希菌原核表达系统进行诱导表达,并用Ni柱对表达的蛋白进行纯化.用IL-4和GM-CSF诱导培养鼠源DCs,用脂多糖(LPS)和L7...

Expression of Brucella ribosomal L7/L12 protein and its effect on mouse derived dendritic cells

In order to study the effect of ribosomal L7/L12 protein of Brucella on the differentiation and maturation of mouse dendritic cells (BM-DCs), the L7/L12 gene was amplified using Brucella S2 vaccine strain as template,and the recombinant plasmid pET30a-L7/L12 was constructed. The recombinant plasmid pET30a-L7/L12 was induced in E. coli prokaryotic expression system, and the expressed protein was purified by Ni column. Mouse-derived DCs was induced by IL-4 and GM-CSF, and the changes of costimulatory molecules and inflammatory factors on the surface of DCs were detected after DCs was stimulated by LPS and L7/L12 proteins. The results showed that the recombinant protein was highly expressed in soluble form under the condition of 1 mmol·L^-1 IPTG and overnight at 16 ℃. The size of the recombinant protein was 18 ku, the purity was more than 93% and had certain reactivity. Flow cytometry showed that the expression of CD40, CD80 and other antigen molecules on the surface of stimulated BM-DCs cells was significantly (P<0.05) higher than that of the blank control group. The results of qPCR showed that the levels of inflammatory cytokines TNF-β, IL-1 β and IL-12 in the control group were significantly (P<0.01) higher than those in the control group. Recombinant L7/L12 protein can stimulate the differentiation and maturation of DCs cells and promote the release of inflammatory factors.