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

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

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/L12蛋白刺激DCs后检测其表面共刺激分子和炎症因子的变化。结果表明,重组蛋白在1 mmol·L^(-1)的IPTG、16℃过夜的条件下以可溶性形式高效表达,其大小为18 ku,纯度达到93%以上并有一定的反应原性。流式细胞仪检测被刺激的BM-DCs细胞表面CD40、CD80等抗原分子的表达显著(P<0.05)高于空白对照组。qPCR结果显示,炎性细胞因子TNF-β、IL-1β、IL-12极显著(P<0.01)高于空白对照组。重组L7/L12蛋白具有刺激DCs细胞分化、成熟和促进炎症因子释放的功能。

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-1βand 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.