关键词: 刚地弓形虫, 肌动蛋白, 基因克隆, 原核表达, 免疫反应性

Abstract: Objective  To clone and express the actin gene of Toxoplasma gondii, and analyze the immunoreactivity of the recombinant protein.  Methods  Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgACT gene was amplified with a pair of specific primers which were designed according to the coding sequence of TgACT gene（Accession No. XM_002369622.1）. The RT-PCR product was cloned into the prokaryotic expression pET-30a（+） vector. The recombinant pET30a-TgACT plasmid was transformed into E. coli DH5α. The positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The correct pET30a-TgACT plasmid was transformed into E. coli BL21（DE3） and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Western blotting assay was performed with anti-poly-histidine tag （anti-His） antibody or rabbit anti-T. gondii serum.  Results  The product of RT-PCR was with 1 100 bp. The recombinant plasmid pET30a-TgACT was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. SDS-PAGE results showed that the target protein was expressed in E. coli BL21（DE3） in the form of inclusion bodies with a rough molecular weight of 49 000. The purified soluble protein was obtained by using denaturation, renaturation and purification. Western blotting revealed that rTgACT can be recognized by anti-His antibody and rabbit anti-T. gondii serum.  Conclusion  The recombinant plasmid pET30a-TgACT has been successfully constructed, and the recombinant protein TgACT is produced in E. coli and maintains specific immunoreactivity.

Key words: Toxoplasma gondii, Actin, Gene cloning, Prokaryotic expression, Immunoreactivity