Abstract: Objective   To identify the protein-protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down.  Methods   The aldolase and actin genes were obtained from cDNA library by PCR amplification，and subcloned respectively into pGEX-4T-1 and pET30a. The fusion protein GST-Aldolase and Actin-His6 were expressed in E. coli upon induction by 1 mmol/L IPTG and then purified with affinity chromatography. Fifteen rats were immunized intradermally with 200 μg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 μg Actin-His6 protein per rat on the 2nd-4th immunizations. Rats were immunized for 4 times with 7 days interval. The serum of rats was collected from heart at the fifth day after the final immunization. Glutathione sepharose beads were incubated with GST-Aldolase protein，then incubated with Actin-His6，and bound proteins were eluted using sample buffer. Eluants were resolved by SDS-PAGE and Western blotting.  Results   The aldolase and actin genes were obtained, and the recombinant plasmid aldolase/pGEX-4T-1，actin/pET30a were successfully constructed. Protein GST-Aldolase and Actin-His6 were expressed and purified in vitro. Serum samples were prepared from rats immunized with protein Actin-His6, and polyclonal antibody was purified with affinity chromatography. SDS-PAGE and Western blotting analysis of products from GST pull-down experiment showed that the protein bands on NC membrane were specifically recognized by anti-Aldolase-His6 and anti-Actin-His6 antibody.   Conclusion  Aldolase interacts with Actin of Toxoplasma gondii.

Key words: Toxoplasma gondii, Aldolase, Actin, GST pull-down, Protein interaction