Cloning And Expression Of The P22 Gene Fragment Of ZS Strains Of Toxoplasma Gondii

Objective: To investigate cloning and expression of the P22 gene fragmant of ZS strains of Toxoplasma gondii.Methods: A 496 bp fragment of the ORF of P22 gene was amplified by PCR from the genomic DNA of ZS strains of Toxoplasma gondii and digested by Pst\ and BgL II. The amplified fragment was inserted into the polylinker of the plasmid pThioHisA,B,C. Then the recombinant plasmid pThioHisA,B,C was transformated into E.coli Top 10. The positive clones Top 10 containing recombinants were idendified by the methods of restrictive digestion and the gene sequencing. The recombinant E.coli were induced by IPTG to express the fusion protein. The expressed and purified products were analyzed by SDS-PAGE and Western-blot . Another 561 bp fragment of the ORF of P22 gene was amplified by PCR, and digested by Pst1 and Xba 1. The amplified fragment was cloned into pBudCFAl to construct the recombinant plasmid pBudCE4.1/p22. Then transfected it into the murine macrophages by using Lipofectamine, the expression of P22-mRNA in the transfected macrophages was identified by RT-PCR. Results:1. The 496 bp fragment of the ORF of P22 gene and a 561 bp fragment were amplified from the genomic DNA of ZS strains of Toxoplasma gondii. Both 496 bp and 561 bp fragments were successfully cloned into the plasmid pThioHisA,B,C and pBudCE 4.1 respectively.2. The recomhinant P22 proteins can be highly expressed as 3xHis Thioredoin fusion proteins in E.coli Top 10. The fusion proteins showed correct bands as analysed by SDS-PAGE and Western-blot with positive serum from rabbit. The recombinant P22 proteins were purified effectively by ProBond?resin .3. The positive clones of the macrophages transfected with pBudCE 4.1/p22 were successfully isolated and the expression of P22-mRNA in the transfected macrophages was identified by RT-PCR.Conclusion: A 496 bp fragment of the gene encoding T gondii P22 surface protein was successfully cloned into the plasmid pThioHisA,B,C and expressed in the E.coli Top 10, and the fusion proteins can be purificated effectively. And the positive clones of the macrophages transfected with pBudCE 4.1/p22 were successfully isolated.