Objective: To clone and express the rhoptry protein 17 (ROP17) gene of RH strain of Toxoplasma gondii, analyze the antigenicity of recombinant protein.
Methods: Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgROP17 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgROP17 gene (GenBank accession No. AM075203.1), the product of RT-PCR was digested with double restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-TgROP17 plasmid was transferred into E. coli DH5alpha and the positive clones were selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The constructed pGEX-6P-1-TgROP17 was transformed into E. coli Rosetta (DE3) and induced with IPTG for expression. The expression products were analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with GST primary antibody and rabbit anti-T. gondii serum was used to confirm the expression of GST-ROP17 and analyze its antigenic properties.
Results: The product of RT-PCR was with 1 850 bp. The recombinant plasmid pGEX-6P-1-TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. A soluble recombinant protein with relative molecular weight of 96,000 was analyzed by SDS-PAGE followed by Coomassie blue staining. The GST tag in GST-ROP17 and the antigenicity of ROP17 were detected efficiently by Western blotting with the GST primary antibody and with the prepared antiserum against T. gondii, respectively.
Conclusion: The recombinant GST-ROP17 protein has been produced in E. coli and shows specific antigenicity.