BACKGROUND: Dense granule antigens (GRA antigens) of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. METHODS: GRA8 complementary DNA (cDNA), encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b(+). Expression of recombinant GRA8 (rGRA8) was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. RESULTS: The cloned gene fragment exhibited complete similarity with the published sequence of gra8 gene by sequence analysis. rGRA8 was expressed in Escherichia coli in fusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRA8 in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. CONCLUSION: The full length soluble rGRA8 was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments.
BACKGROUND: Dense granule antigens (GRA antigens) of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. METHODS: GRA8 complementary DNA (cDNA), encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b(+). Expression of recombinant GRA8 (rGRA8) was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. RESULTS: The cloned gene fragment exhibited complete similarity with the published sequence of gra8 gene by sequence analysis. rGRA8 was expressed in Escherichia coli in fusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRA8 in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. CONCLUSION: The full length soluble rGRA8 was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments.