Analyzation Of The Protein Interactions Between The Dense Granules Antige7(GRA7) Of T.Gondii And Host Components

Objective: To clone and express the GRA7gene of Toxoplasma gondii strain RH inE.coli to produce GST-His-GRA7recombinant fusion protein, evaluate theantigenicities of the GRA7recombinant protein, and analyze the protein interactionsbetween the dense granules antige7(GRA7) of T. gondii and host components, whichlay the foundation of researching the infect-immune mechanism and theropy ofToxoplasmsis.Methods: In vitro，Hela cell lines were used to culture Toxoplasma tachyzoite，separate and purificate tachyzoite by the means of density gradient centrifugation andPercoll cell separation solution. The cDNA were synthetized from the total RNA ofToxoplasma gondii. The total length of GRA7was amplified using PCR, and ligatedinto the pET-41Ek/LIC vector by direct nucleotide sequence recombination. Therecombinant fusion protein with His label was induced in E.coli strain RosettaTM(DE3) pLysS.The prokaryotic GRA7recombinant protein with His label wasproduced and purified by Ni-NTA spin columns, then analyzed by SDS-PAGE andWestern blotting. Mice were immunized with purified GRA7recombinant protein todevelope antibodies. Fusion protein was recognized by immune mice and humanserum. Stimulate and differentiate the THP-1cell using Toxoplasma cultured byTHP-1cell，collecte cell lysate to conduct GST pull-down experiment, and use liquidchromatography and protein substances crawl by LC-MS/MS for proteinidentification. Clone and express in E.coli to produce identified carbonic anhydraserecombinant fusion protein which was used to make immune mice serum, it couldconduct Co-IP to identify the interaction between these proteins. The impaction oftachyzoite growth was observed by pcDNA3.1-CA transient transfection in THP-1cell.Results: The GRA7recombinant protein was verified to have relatively high antigenicities using immune hybridizations of recombinant proteins and patientserums, which indicated that they can be used in the clinical diagnosis of currentinfection. T. gondii GRA7secreted proteins significantly interacted with the hostcarbonic acid metabolism enzymes, and the over-expression of host carbonicanhydrase (CA) improved the T. gondii propagation speed significantly, but thenumber was almost same.Conculsion: The Toxoplasma gondii GRA7proteins with good immunogenicity thatcan provides references to clinical detection. Host carbonic acid metabolism enzymescaptured by the GRA7can directly or indirectly influence the intracellularpropagation of T. gondii. It was supposed that the CA enzyme was related with theformation and stability of parasitophorous vacuole.