| SAG2 is an important antigen on the surface of the tachyzoite of Toxoplasma gondii, which is significant for prevent and detect toxoplasmosis.At first,SAG2 gene was respectively inserted into plasmid pPIC9k,pGEX-4T-2 and pET28b to construct the recombinant plasmid pPIC9k-SAG2,pGEX-4T-SAG2 and pET28b-SAG2.After transferred into the GS115 and E.coli BL21(DE) cells, the recombinant plasmids were expressed, and the proteins around 15KD,41KD and 20KD were gotten. The output of GST-SAG2 protein is the most by the results of SDS-PAGE. Then GST-SAG2 was purified with GSTrap FF by AKTA purifer system. Western-blotting shows it can react with rabbit-anti Toxoplasma gondii serum. The optimization experiments indicated that highest yield can be gotten by 2×YTA, 0.1mM IPTG, 3 hours, 220rpm and at 30℃. The yield is more than 25% of all of expression proteins.Using the target protein as the antigen, the optimum condition of ELISA was determined. The optimal dilution of GST-SAG2 for coating of plate was 1:100 (0.25ug/ml), the optimal dilution of serum sample was 1:200 and the working cconcentration of HRP-labeled antibody was 1:2000 were determined. Following the determination of condition of ELISA, the threshold value of ELISA was 0.214.20 control positive/negative serums were detected with this method and the kit respectively. It was found that the coincidence of ELISA were 100%. The results of the cross-reaction revealed that the ELISA by the purified SAG2 had good specificity for the detection of SAG2 antibody in serum. The study have developed an assay, which provides an available technique for immune detection and serological survey of Toxoplasma gondii.
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