[Interaction between Toxoplasma GRA7 dense granule protein and the protein from host macrophages].

OBJECTIVE: To identify the protein from host macrophages which interacted with GRA7 dense granule protein of Toxoplasma gondii, and reveal the relationship between protein interaction and infection process. METHODS: The recombinant GRA7 protein with N-terminal GST tag were used as a bait in in vitro GST Pull-down experiment, the proteins of THP-1 monocytic macrophage cell line were captured and identified by LC-MS/MS proteomics method. The in vivo protein interaction was verified by Co-IP experiment The overexpression of the target host protein by pcDNA3.1 (+) vector in THP-1 macrophage was further used to analyze the relationship between protein interaction and infection process. RESULTS: The captured THP-1 cell protein was about Mr 29000, which was identified as human carbonic anhydrase 1 (hCA1). The significant in vivo protein-protein interaction between GRA7 and hCA1 was verified by Co-IP assay. The overexpression of hCA1 gene in THP-1 macrophage induced a higher propagation speed of Tgondii and the formation of the parasitophorous vacuole, but did nmt influence the number of the parasite. CONCLUSION: There is a significant protein interaction between Toxoplasma GRA7 dense granule protein and hCA1 enzyme from host macrophages, which is positively related with the propagation speed of T. gondii.

[Separation and identification of the surface and secreted proteins in Toxoplasma gondii by biotin labeling and proteomics methods].

OBJECTIVE: To separate and identify the surface proteins and secreted proteins of Toxoplasma gondii tachyzoites of RH strain. METHODS: T. gondii tachyzoites were cultured in Vero cells, and purified by filtration and Percoll cell separation solution. The biotin-labeled tachyzoites were lysed, and the surface and secreted proteins were separated by NeutrAvidin agarose beads. After condensation and SDS-PAGE, the protein were collected, digested and identified by LC/MS-MS. RESULTS: A total of 785 T. gondii proteins were identified, 81 (10.3%) of which were originally annotated as the surface or secreted proteins. Among the highly-expressed (PSM>10) 65 proteins, 43 (66%) were originally annotated as surface or secreted proteins, while the others were predicted unknown proteins. CONCLUSION: The surface and secreted proteins of T. gondii are separated by biotin labeling and avidin chromatography, among which some potential new surface or secreted proteins of T. gondii are identified.