布鲁杆菌Cu-Zn SOD和GroEL的克隆表达及细胞免疫特性比较分析  

Expression and immunologic features of Cu-Zn SOD and GroEL proteins of Brucella melitensis

作　　者：于爽[1] 徐杰[2] 付思美[2] 王玉飞[2] 白耀霞[1] 钟志军[3] 陈燕芬[2] 王同坤[2] 杨毅[2] 杜昕颖[2] 汪舟佳[2] 李竞[1] 黄留玉[2] 陈泽良[2] 

机构地区：[1]西南大学药学院,重庆400715 [2]军事医学科学院疾病预防控制所,北京100071 [3]四川农业大学动物医学院,四川雅安625014

出　　处：《解放军医学杂志》2010年第9期1102-1105,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(30901071);传染病重大专项基金(2008ZX10004-015;2009ZX10004-103);国家"863"计划项目(2007AA02Z412)

摘　　要：目的分析比较布鲁杆菌两种外膜蛋白Cu-Zn SOD和GroEL的细胞免疫特性。方法以布鲁杆菌16M基因组为模板,PCR扩增Cu-Zn SOD和GroEL蛋白的基因序列。采用Gateway方法构建表达载体,在大肠埃希菌中诱导表达并纯化,用获得的纯化蛋白刺激减毒活疫苗S19免疫小鼠的脾细胞,并测定细胞上清中IFN-γ的水平。结果成功克隆表达了Cu-Zn SOD和GroEL蛋白,用这两种蛋白刺激S19免疫小鼠脾细胞后,细胞上清中的IFN-γ水平明显升高,并且GroEL刺激后的IFN-γ水平高于Cu-Zn SOD刺激后的IFN-γ水平。结论 GroEL和Cu-Zn SOD蛋白均具有细胞免疫反应特性,且GroEL的细胞免疫反应特性强于Cu-ZnSOD。Objective Outer membrane protein Cu-Zn SOD and GroEL of Brucella may induce immune response in the host.The cell immunity of the two outer membrane proteins was compared for screening an antigen with higher cell-mediated immunity.Methods By using genomic DNA of Brucella melitensis 16Mas template,the coding sequences of the two genes were amplified,and then cloned into expression plasmid by gateway technology.The proteins were induced for expression in E.coli and purified by affinity chromatography. The purified proteins were then used to stimulate the spleen cells of mice immunized with S19,a live attenuated vaccine strain.The secretion of IFN-γwas evaluated by detection of IFN-γin supernatant of the stimulated cells.Results The genes of the two outer membrane proteins Cu-Zn SOD and GroEL were successfully cloned and expressed in E.coli.Both of them induced higher levels of IFN-γin the stimulated supernatants.Comparison of the two proteins showed that the IFN-γlevel of GroEL stimulated cells was higher than that of Cu-Zn SOD,indicating that both proteins could induce cell-mediated immunity in mice,and furthermore,the GroEL induced higher levels of cell-mediated immunity than Cu-Zn SOD did.Conclusion Both of the two outer membrane proteins can induce cell-mediated immunity in mice,but GroEL may induce higher levels of cell-mediated immunity than Cu-Zn SOD,implying that it is more appropriate to use GroEL as a sub-unit vaccine candidate.

关 键 词：布鲁杆菌属 GATEWAY技术 免疫 细胞 

分 类 号：R392.111[医药卫生—免疫学]