Abstract: Objective:To discuss the prokaryotic expression system and purification conditions of Toxoplasma gondii profilin-like protein (TgPRF), and to provide basis for the study on anti-tumor immuno-adjuvant. Methods:The coding region of TgPRF gene was amplified with a pair of specific primers which were designed according to the cDNA oftachyzoites of Toxoplasma gondii RH strain. The PCR products were cloned into the pET-28a(+) vector after double enzyme digestion. The recombinant pET28a(+)-TgPRF plasmid was transformed into E.coli DH5α cells. The positive clones were selected by the double restrictive enzyme digestion and sequencing. The correct pET28a (+) -TgPRF plasmid was transformed into E.coli BL21(DE3) and induced for 4 h by IPTG. The expression of recombinant TgPRF protein was analyzed by SDS-PAGE method; the expression of recombinant protein His-profilin was detected by Western blotting method. Results:The length of product of PCR was 492 bp. The recombinant plasmid pET28a-TgPRF was confirmed by double restriction enzyme digestion and sequencing. The SDS-PAGE results showed that the target protein was expressed in E.coli BL21 (DE3) in bacteria supernatant. The purified TgPRF protein was obtained by Ni-NTA agarose gel column chromatography with the purity>90%. The Western blotting results revealed that the recombinant TgPRF protein could be recognized by Anti-His antibody. Conclusion:The recombinant plasmid pET28a-TgPRF is successfully constructed, and the TgPRF protein is obtained with the soluble prokaryotic expression.

Key words: Toxoplasma gondii, profilin, prokaryotic expression, protein purification