【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。更多还原

【Abstract】 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.更多还原