Molecular cloning and expresssion of T.gondii surface antigen SAG1 gene in E.coli

Objective To clone and express surface antigen SAG1 gene from Toxoplasma gondii RH strain.Methods T.gondii RH strain tachyzoites,which maintained by mouse passage,were harvested from ascites of mice and total RNA was prepared. A pair of primers were designed and synthesized based on the sequence of SAG1 cDNA. A specific fragment of SAG1 gene was obtained by RT-PCR amplification. The RT-PCR products were ligated to pGEM-T. The EcoRI/HindⅢ restricted fragments,confirmed by PCR and EcoRI/HindⅢ digestion,were cloned into expression vector pET28a and the recombinants were transformed into E.coli BL 21.Fusion expression was induced by beta-D-thiogalactosidase(IPTG) and identified by western blot with anti-Toxoplasma sera.Results The molecular size of SAG1 was 1 011 bp,which was identical to the previous report cloned from the parasites of intestinal epithelial stage in cat. High expression was obtained in pET28a/SAG1/E.coli BL 21when confirmed by western blot.Conclusions The recombinant construction of SAG1 is generated and expression is induced.The recombinant P30 has a specific immunoactivity and potentiality in immunological diagnosis.