弓形虫表面抗原P35重组蛋白的表达、纯化及鉴定

目的 构建弓形虫R0H株表面抗原P35基因重组表达质粒P35／pGEX-2T，并在大肠杆菌JMl09中进行表达、纯化及鉴定。方法 采用逆转录-聚合酶链反应(RT-PCR)技术从弓形虫CDNA库中扩增出编码P35抗原的基因片段，克隆入表达载体pGEX-2T，并在大肠杆菌JMl09中融合表达，经GSTmp^TM亲和层析柱纯化出P35重组融合蛋白，以SDS-PAGE电泳鉴定纯度、Western-blot鉴定抗原性。结果成功构建了重组表达质粒P35／pGEX-2T，融合表达产物的分子质量约为70kDa；该蛋白抗原能为谷胱甘肽S转移酶(GST)单克隆抗体及弓形虫感染的兔血清所识别。结论弓形虫表面抗原P35在大肠杆菌中有效表达，纯化蛋白有良好的免疫活性。

Expression, purification and characterization of P35 recombinant antigen of Toxoplasma gondii

Objective Expression,purification and characterization of P35 recombinant antigen of Toxoplasma gondii in E.coli JM109. Methods The gene encoding P35 surface antigen was amplified by RT-PCR,from a Toxoplasma gondii cDNA libray,and cloned into plasmid pGEX-2TK to construct a recombinant plasmid P35/pGEX,Then transformed it into E.coli JM109.The positive recombinant clone was induced by IPTG to express target fusion protein GST-p35 which was easy to be purified with GSTrap TM column and Trombin Protease.The purified product was analyzed by SDS-PAGE and Western-blot. Results The fusion GST-P35 protein was about 54 kDa and P35 recombinant antigen protein about 28 kDa respectively.The purified GST-P35 protein can be recognized by anti-GST antibody and the serums of infected rabbits by Western-blot. Conclusion The recombinant plasmid P35/pGEX was constructed and expressed in E.coli JM109,and the purified fusion protein GST-P35a and P35 recombinant protein have good antigenicity.