[In vitro Effect of Hypericin against Toxoplasma gondii Tachyzoites].

Objective: To investigate the killing effect of hypericin on tachyzoites of Toxoplasma gondii RH strain in vitro. Methods: Normal saline （group A） and different concentrations of hypericin (5 µg/ml, group B; 50 µg/ml, group C; 500 µg/ml, group D） were added to T. gondii tachyzoites in 24-well plate（1×10(6)/well）. The tachyzoites were harvested after 2, 4 and 6 h, and underwent the following treatment: trypan blue staining to calculate the dyeing rate, Giemsa staining to observe the morphological and structural alterations of tachyzoites, and transmission electron microscopy to observe the ultrastructure of tachyzoites. In addition, flow cytometry was performed to calculate the survival rate of YFP-carrying Toxoplasma with the same treatment. Results: The trypan blue dyeing rate at 2 h after treatment in groups B, C and D was（11.0±3.6）%, （25.0±6.3）% and（40.0±2.7）% respectively, with a significant difference of group D versus B and C （P<0.01）, and groups C and D versus group A ［（6.0±3.0）%）］. The dyeing rate at 4 h and 6 h in group D was（97.0±2.0）% and （98.0±1.7）%, respectively, both significantly higher than that of groups C ［（30.0±7.2）%, （42.7±5.5）%ï¼½, B ［（20.0±3.0）%, （34.0±6.6）%ï¼½ and A ［（10.0±1.0）%, （19.3±4.9）%］（P<0.01）. Giemsa staining showed gradual end swelling and necrosis of tachyzoites with increased treatment duration and dosage. Transmission electron microscopy showed swelling of worm body, gap between cell membrane and matrix, increase and enlargement of vacuoles inside worm body, disruption of cell membrane, and dissolving of inner structures, with increased treatment duration. Flow cytometry showed significant difference of tachyzoite survival rate at 2, 4 and 6 h after hypericin treatment with that of the control group（P<0.01）. The survival rate of group C at 2 h after hypericin treatment was（7.9±1.9）%, significantly lower than that of groups B ［（38.1±5.5）%ï¼½ and A ［（81.8±6.0）%ï¼½ （P<0.01）. No tachyzoite was found to survive in group D at 2 h and in group C at 4 h. The survival rate of group B at 4 and 6 h after hypericin treatment was（14.3±7.9）% and （1.4±1.8）%, respectively, both significantly lower than that of group A［（73.8±11.3）% and（64.1±14.4）%, respectivelyï¼½ （P<0.01）. Conclusion: Hypericin has a remarkable killing effect on T. gondii tachyzoites, and the efficacy positively correlates with the dose and treatment duration.

[Cloning, expression and identification of gametocyte specific protein Pfgdv1 of Plasmodium falciparum].

OBJECTIVE: To clone a gametocyte specific protein Pfgdv1 of Plasmodium falciparum, express and identify recombinant Pfgdvl protein in vitro. METHODS: PCR was performed to amplify Pfgdv1 from P. falciparum DNA which was got from the patient who was infected with P. falciparum, and the PCR product was inserted into pET28a (+) vector. pET28a-Pfgdv1 recombinant plasmid was constructed and transformed into E. coli host BL21 (DE3+). IPTG was used to induce the recombinant Pfgdv1 protein fused with His tag, and the protein was purified by His-NTA affinity chromatography. The recombinant protein was identified by SDS-PAGE and Western blotting. RESULTS: The PCR product of Pfgdv1 gene was about 1.65 kb, meeting the expectation of predicted fragment size. The recombinant protein was about 67 kDa, which could be recognized by His-Tag monoclonal antibody. CONCLUSION: The Pfgdv1 gene of P. falciparum is successfully cloned, and the recombinant Pfgdv1 protein is expressed, thereby providing an opportunity for further study on transmission blocking vaccine.