Sugi Tatsuki, Masatani Tatsunori, Murakoshi Fumi, Kawazu Shin-ichiro, Kato Kentaro
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan; Transboundary Animal Diseases Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima 890-0065, Japan.
Anal Biochem. 2014 Nov 1;464:9-11. doi: 10.1016/j.ab.2014.06.018. Epub 2014 Jun 30.
Toxoplasma gondii can differentiate into tachyzoites or bradyzoites. To accelerate the investigation of bradyzoite differentiation mechanisms, we constructed a reporter parasite, PLK/DLUC_1C9, for a high-throughput assay. PLK/DLUC_1C9 expressed firefly luciferase under the bradyzoite-specific BAG1 promoter. Firefly luciferase activity was detected with a minimum of 10(2) parasites induced by pH 8.1. To normalize bradyzoite differentiation, PLK/DLUC_1C9 expressed Renilla luciferase under the parasite's α-tubulin promoter. Renilla luciferase activity was detected with at least 10(2) parasites. By using PLK/DLUC_1C9 with this 96-well format screening system, we found that the protein kinase inhibitor analogs, bumped kinase inhibitors 1NM-PP1, 3MB-PP1, and 3BrB-PP1, had bradyzoite-inducing effects.
刚地弓形虫可分化为速殖子或缓殖子。为加速对缓殖子分化机制的研究，我们构建了一种用于高通量检测的报告寄生虫PLK/DLUC_1C9。PLK/DLUC_1C9在缓殖子特异性BAG1启动子的驱动下表达萤火虫荧光素酶。在pH 8.1诱导的至少10²个寄生虫中可检测到萤火虫荧光素酶活性。为使缓殖子分化标准化，PLK/DLUC_1C9在寄生虫的α-微管蛋白启动子的驱动下表达海肾荧光素酶。在至少10²个寄生虫中可检测到海肾荧光素酶活性。通过使用PLK/DLUC_1C9和这种96孔板形式的筛选系统，我们发现蛋白激酶抑制剂类似物，即撞击激酶抑制剂1NM-PP1、3MB-PP1和3BrB-PP1，具有诱导缓殖子形成的作用。
