[Protein interaction site of Toxoplasma gondii microneme protein 6 and aldolase determined by site-directed mutagenesis].

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/N 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.

Effect of arginine vasopressin on the behavioral activity in the behavior despair depression rat model.

Arginine vasopressin (AVP), a nonapeptide posterior hormone of the pituitary, is mainly synthesized and secreted in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). Large numbers of studies have reported that AVP plays a role in depression. The present study was to investigate by which level, brain or periphery, AVP affects the behavioral activity in the behavior despair depression rat model. The results showed that (1) either forced swimming or tail suspension significantly increased AVP concentration not only in the brain (PVN, SON, frontal of cortex, hippocampus, amygdala, lumber spinal cord) but also in the periphery (posterior pituitary and serum); (2) intraventricular injection (icv) of AVP decreased the animal immobility time, whereas V1 receptor antagonist d(CH2)5Tyr(Me)AVP (icv) increased the animal immobility time in a dose-dependent manner not only in FST but also in TST, but the V2 receptor antagonist d(CH2)5[D-Ile, Ile, Ala-NH9]AVP did not change the animal immobility time in FST or TST; (3) V1, not V2 receptor antagonist could inhibit the animal immobility time decrease induced by AVP (icv); (4) neither AVP nor its receptor antagonist (including V1 and V2 receptor antagonist) influenced the animal immobility time in both FST and TST. The data suggested that AVP in the brain rather than the periphery played a role in the behavior despair depression by V1, not V2 receptors, which behavior despair might have a positive feedback effect on central AVP and blood AVP might have a negative feedback on central AVP in the depressive process.

[Expression of aldolase of Toxoplasma gondii and preparation of the polyclonal antibodies against this protein].

AIM: To produce the aldolase protein and its polyclonal antibody. METHODS: Aldolase gene was obtained from cDNA library by PCR amplification and subcloned to vector pET30a. The recombinant protein aldolase-His(6); was expressed in E.coli upon IPTG induction and then purified with affinity chromatography. The purified protein mixed with adjuvant was used to immunize SD rats to produce the polyclonal antibodies. RESULTS: The recombinant plasmid aldolase/pET30a was constructed successfully and expressed as a fusion protein aldolase-His(6);; Polyclonal antibody against aldolase-His(6); was obtained from rat, and antibody titer was 1:4 000. CONCLUSION: The purified protein aldolase-His(6); and its polyclonal antibodies were obtained, which may provide the foundation for the further studies on the function of aldolase.

[Protein interaction between aldolase and actin of Toxoplasma gondii by GST pull-down].

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 microg Actin-His6 protein per rat at first time to produce the polyclonal antibodies. Then 100 microg 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.