细粒棘球蚴重组14-3-3基因的表达、纯化及免疫学鉴定

Expression,purification and immunologic identification of the recombinant 14-3-3 gene from Echiococcus granulosus(Chinese mainland strain)

目的构建细粒棘球蚴14-3-3基因的重组质粒并原核表达、纯化该重组蛋白,对其免疫学特性进行初步鉴定。方法从重组质粒pGEM-T/Eg14-3-3中获取14-3-3基因,亚克隆于表达载体pET28a构建基因工程菌株,并表达、纯化重组蛋白,经Western blot、ELISA对该蛋白的免疫学特性进行初步研究。结果成功构建含目的片段14-3-3的基因工程菌株;ELISA检测显示,用表达、纯化的重组蛋白免疫小鼠,诱导产生了特异性抗体,Western blot鉴定该抗体能识别重组抗原及原头蚴、囊液、囊壁抗原。结论构建的pET28a/Eg 14-3-3菌株能高效表达14-3-3蛋白,初步鉴定该重组蛋白具有较好的抗原性和免疫原性。

Objective To construct Eg14-3-3 gene and express prokaryotically it, to purify and identify the immunogenicity of recombinant 14-3-3 protein. Methods Egl4 3 3 gene was attained from pGEM T/Eg14-3-3 recombinant plasmid and subcloned into high-level expressed vector pET28a. Eg14-3-3/pET28a/BL21plys was constructed. Western blot and ELISA were used to identify the immunogenicity of the recombinant protein. Results Egl4 3 3/pET28a/BL21plys recombinant plasmid was constructed. ELISA indicated that 14-3 3-immunized mice could produce the specific antibody; Western blot analysis showed that recombinant protein and protoscolex, cystic fluid and cystic wall of Echiococcus granulosus. could be recognized by this antibody. Conclusion pET28a/Eg14-3-3 recombinant plasmid is constructed and expressed efficiently. The antigenicity and immunogenicity of Eg14-3-3 protein is identified.