关键词: 刚地弓形虫, 微线体蛋白6, 醛缩酶, 定点突变技术, 蛋白作用位点

Abstract: Objective  To identify the protein interaction site of Toxoplasma gondii microneme protein 6 （MIC6） and aldolase by using site-directed mutagenesis.  Methods  Based on Toxoplasma gondii MIC6 gene sequence （GenBank Accession No. AF110270）, the specific primers were designed. Tryptophan（W）-348 of MIC6 C terminus （MIC6C） was mutated to valine（V） via site-directed mutagenesis. MIC6C W/V gene was obtained from cDNA library by PCR amplification and subcloned into pGEX-4T-1. The mutant protein GST-MIC6C W/V was expressed in E. coli, induced by 0.8 mmol/L IPTG, and purified by affinity chromatography. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with T. gondii tachyzoites lysate, and bound proteins were eluted using sample buffer. Bound products were resolved by SDS-PAGE and Western blotting. Glutathione sepharose beads were incubated with GST-MIC6C W/V and GST-MIC6C, respectively, and then incubated with aldolase-His6. After incubation, the resin was washed and subjected to SDS-PAGE.  Results  The MIC6C W/V gene was obtained, and the recombinant plasmid MIC6C W/V/pGEX-4T-1 was successfully constructed. The mutant protein GST-MIC6C W/V was expressed and purified in vitro. SDS-PAGE analysis indicated that GST-MIC6C was co-precipitated with aldolase from T. gondii tachyzoites lysate or aldolase-His6, whereas GST-MIC6C W/V failed to precipitate aldolase from T. gondii tachyzoites lysate or aldolase-His6. Western blotting analysis using anti-aldolase antibody indicated that GST-MIC6C could pull-down aldolase from T. gondii tachyzoites lysate.  Conclusion  Tryptophan （W348） was the interaction site of MIC6 and aldolase in T. gondii.

Key words: Toxoplasma gondii；Microneme protein 6（MIC6）；Aldolase；Site-directed mutagenesis;      , Protein interaction site