Molecular Cloning, Expression And Identification Of TgMIC10 Gene From Toxoplasma Gondii In Vitro

Objective: To set up a way of quickly and accurately detecting toxoplasmosis by use of recombinant protein, the micronal seceted protein TgMIC10 was amplified from Toxoplasma gondii (RH strain) and subcoloned into the expression vector pBK-CMV. Methods: The specific primers were designed and synthesized. The DNA fragment encoding T. gondii TgMICIO was 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 TgMIC10 was subcloned into pBK-CMV expression vector and the recombinant plasmid was transformed into E. coli BL21, IPTG was added to induce expression of fused TgMIC10. The expression was identified by Western blot. Results: The size of pGEM-T-TgMIC10, pBK-CMV-TgMIC10 digested with EcoR I and Xba I was identical to the length of the PCR generated products. DNA sequencing indicated that the TgMICIO gene has an open reading frame of 597bp, which encodes 198 amino acids with an approximate molecular weight of 23kDa and has high homology with the TgMICIO gene submitted to GenBank (AF293654). It indicated that TgMIC10 gene was successfully cloned and subcloned into the vectors. With the positive clones induced by IPTG, a fusion protein was expressed in E.coli BL21. The fusion protein could be recognized by the sera of rabbit, infected with T. gondii tachyzoites.