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首页> 外文期刊>Parasitology >Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins.
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Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins.

机译：通过阶段特异性双重荧光蛋白的表达可视化弓形虫阶段转化。

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摘要

To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7.0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.
机译：为了识别弓形虫在活宿主细胞中的速殖子和缓殖子之间的阶段转换，构建了表达阶段特异性红色和绿色荧光的转基因弓形虫品系。分别在速殖子特异性SAG1和缓殖子特异性BAG1启动子的控制下，用编码红色荧光蛋白（DsRed Express）和绿色荧光蛋白（GFP）的基因稳定转化了弓形虫PLK菌株速殖子。所得的转基因寄生虫称为PLK / DUAL。当在pH 7.0的培养基中培养PLK / DUAL时，PLK / DUAL的沸石显示红色荧光，但未观察到可检测水平的绿色荧光。 PLK / DUAL zoites与抗SAG1抗体反应，但与抗BAG1抗血清反应。当在高pH条件下或在存在p38 MAPK抑制剂SB202190的情况下培养PLK / DUAL时，少量的动物卵石表达绿色荧光，并且为BAG1阳性。 C57BL / 6J小鼠感染了PLK / DUAL速殖子。在急性和活化阶段，动物体表达红色荧光。但是，无法检测到绿色荧光。相反，潜在的囊肿表达绿色荧光。 PLK / DUAL的阶段特异性双重荧光有助于鉴定活细胞中的寄生虫阶段，其优点是不需要固定或免疫染色。

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