Molecular Cloning And Expression Of Signaling Protein 14-3-3 And SAG1 Gene From Toxoplasma Gondii And Its Primary Use In Immunodiagnosis Of Toxoplasmosis

Objective: To generate the signaling protein 14-3-3 of T. gondii (Toxol4-3-3) andsurface antigen SAG1 following inserts ligation, cell transformation, and induced expression for quick and accurate immunodiagnosis of toxoplasmosis. Methods: The specific primers were designed and synthesized. The cDNA fragment encoding T. gondii signaling protein 14-3-3 and surface antigen SAG1 were amplified by RT-PCR from RNA of RH strain tachyzoites. The PCR products were cloned into pGEM-T vector and recombinants were screened by PCR, enzyme digestion and sequencing. Then the 14-3-3 gene and SAG1 were subcloned into pET28a expression vector, and-E. coli BL21(DE3) was used for transformation. The expression of Toxo 14-3-3 and SAG1were induced by IPTG and identified by Western blot. In order to evaluate rToxo14-3-3 and rSAGl, we use them to detect positive sera from Toxoplasma-infected cases. Results: The size of pGEM-T,Toxo14-3-3, pET28a, Toxol4-3-3 digested with EcoR I and Xho I were all the same in length as the Toxol4-3-3-PCR product; pGEM-T ,SAGl, pET28a,SAGl digested with EcoR I and Hind III were all the same in length as the SAG1-PCR product . DNA sequencing indicated the Toxol4-3-3 gene had an open reading frame of 803bp, which encodes 266 amino acid with a molecular standard of 30.7 kDa identical to Toxo 14-3-3 gene submitted to GenBank (ABO 12775); SAGl has an open reading frame of 1011bp, which encodes 336 amino acid with a molecular standard of 34.81 kDa identical to SAGl gene submitted to GenBank (S76248). Conclusion: Toxo 14-3 -3 and SAGl gene were successfully cloned-and expressed in E.coli BL21.The fusion proteins have been confirmed by serai recognition of rabbit infected with tachyzoites. After simple purification procedure, the expressed product was identified by western blot. These results of serai detection test indicate that the ELISA using rToxo 14-3-3 can be useful for effective and reliable serodiagnosis of toxoplasmosis.