布鲁菌抗原的快速克隆与高效表达  

Rapid Cloning and High Expression of Brucella melitensis Antigens

作　　者：徐杰[1,2] 于爽[2] 付思美[2] 钟志军[2] 王玉飞[2] 曲勍[2] 汪舟佳[2] 杜昕颖[2] 孙宏迪[2] 白耀霞[2] 黄留玉[2] 高岚[1] 陈泽良[2] 

机构地区：[1]兰州大学发育生物学研究所,甘肃兰州730000 [2]军事医学科学院疾病预防控制所,北京100071

出　　处：《生物技术通讯》2010年第3期323-327,共5页Letters in Biotechnology

基　　金：国家"十一五"重大传染病专项(2008ZX10004-015);国家高技术研究发展计划(2007AA02Z412);国家自然科学基金(30970312)

摘　　要：目的:通过Gateway技术构建布鲁菌抗原表达载体,并筛选出高效可溶性表达载体。方法:以山羊布鲁菌16M株染色体DNA为模板,扩增4个布鲁菌抗原基因BMEI2002、BMEI1069、BMEI1483和BMEI0748,利用GatewayBP反应将基因克隆到入门载体pDONR201中,构建重组质粒,然后用Gateway LR反应将基因重组到3种表达载体(pDEST17、pHXGWA、pHGGWA)中,构建相应的重组表达质粒,将重组质粒转化大肠杆菌ER2566(DE3)并诱导表达,分析利用3个不同载体所表达蛋白的表达量及表达形式。结果:利用BP反应构建了4个基因的重组质粒,用LR反应将这些基因分别克隆到表达载体,构建得到了相应的表达载体;诱导表达后的可溶性分析显示,含6×His和TRX标签的pHXGWA所表达的蛋白在表达量和可溶性方面均优于pDEST17和pHGGWA。结论:通过Gateway技术实现了布鲁菌抗原的快速克隆,筛选到的pHXGWA可作为后续大规模克隆表达载体,为布鲁菌抗原的大规模克隆表达和保护性抗原的筛选奠定了基础。Objective：To construct Brucella antigens expression plasmids by the Gateway technology,and screen the plasmid for high and soluble expression of Brucella antigens from them.Methods：The four antigen genes BMEI2002,BMEI1069,BMEI1483 and BMEI0748 were amplified from the genomic DNA of Brucella melitensis 16M strain template,and BP cloned into entry plasmid pDONR201,then,these genes were sub-cloned into three different expression plasmids pDEST17,pHXGWA and pHGGWA respectively.The total 12 recombinant plasmids were transformed into E.coli ER2566（DE3） respectively,and expression forms of the recombinant proteins were ana-lyzed by SDS-PAGE.Results：The four genes were successfully amplified and BP cloned into entry vectors,and then sub-cloned into expression plasmids.The results of SDS-PAGE showed that plasmid pHXGWA with the tags of 6×His and TRX was better than the other two plasmids in both expression level and solubility of the proteins.Conclusion：The plasmid pHXGWA was determined to be used for large-scale expression and for screening of protective antigens further.

关 键 词：GATEWAY 布鲁菌 蛋白表达 可溶性分析 

分 类 号：Q78[生物学—分子生物学]