日本血吸虫(中国大陆株)信号蛋白14-3-3在原核细胞的高效融合表达和特性鉴定

Identification and expression of signaling protein 14-3-3 of Schistosoma japonicum(Chinese mainland strain) in prokaryotic cells

目的 构建重组质粒pET2 8a -Sj14 - 3- 3,并在大肠杆菌中表达 ,检测表达产物的免疫活性。 方法 用亚克隆技术把Sj14 - 3- 3基因克隆至 pET2 8aT7启动子下游 ,转化大肠杆菌DH5α和BL2 1感受态细胞 ,经IPTG诱导表达 ,SDS -PAGE和Westernblot分析。结果 获得pET2 8a -Sj14 - 3- 3重组表达载体 ,SDS -PAGE和Westernblot显示Sj14 - 3- 3基因在 pET2 8a中表达的融合蛋白约为 32 4kDa ,与天然 14 - 3- 3蛋白具有相同的免疫活性。 结论 日本血吸虫信号蛋白14 - 3- 3在原核细胞中得以高效表达 ,表达产物具有免疫活性 。

Aim To construct a recombinant plasmid pET28a-Sj14-3-3,and detect the immunoactivity of the expression in E coli Methods Subclone the Sj14-3-3 encoding gene into the downstream of the T7 promoter of vector pET28a,transform the recombinant plasmid into competent cells of E coli DH5α and BL21,and induced by IPTG The cell lysates were analysed with SDS-PAGE followed by Western blot Results The recombinant expression plasmid pET28a-Sj14-3-3 was successfully constructed SDS-PAGE and Western blot showed a fusion protein with the molecular weight of 32 4KDa and the expressed product had the same immunoactivity as native 14-3-3 protein Conclusion Signaling 14-3-3 protein of Schistosoma japonicum(Chinese mainland strain) was high expressed in prokaryotic cell and identified by anti-14-3-3ε immunogen The primary work provided a new approach to molecular candidate vaccine and signal transduction of Schistosome