High expression and purification of immunogenic recombinant T.gondii SAG1 gene in E.coli and its preliminary application in T.gondii infection detection

Objective To investigate the expression of truncated T.gondii surface antigen SAG1 in E．coli and the detection of T.gondii infection with this recombinant antigen. Method The truncated SAG1 was subcloned into a prokaryotic expression vector pET-30a(+), and the recombinant plasmid was identified by enzyme digestion and the reading frame was confirmed by sequence analysis. A bacterium BL21(DE3) was transformed with the recombinant plasmid and the recombinant product expressed in E．coli after a 3-hour induction with isopropyl beta-D-thiogalactosidase (IPTG), and the immunogenicity of the purified and refolded SAG1 (rSAG1) was tested with Western blot and ELISA. Results The truncated SAG1 of T．gondii was successfully subcloned and highly expressed in E．coli, and the expression product accounted for 31.58% of the total protein of cell lysate. After simple purification and refolding procedures, rSAG1 could be recognized by human toxoplasma-infective serum. The ELISA kits constructed by the rSAG1 were demonstrated to be highly sensitive and specific for the detection of Toxoplasma infection, almost comparablewith immunoblotting method. Conclusion The truncated SAG1 was highly expressed in E．coli as a fusion protein and the recombinant antigen is immunogenic after simple purification and refolding procedures. The ELISA kit constructed with rSAG1 can be used to detect Toxoplasma infection.