利用CRISPR/Cas9构建约氏疟原虫有性阶段的荧光报告虫株
首发时间:2017-05-04
摘要:利用实验室在疟原虫中建立的CRISPR/Cas9系统,本研究将疟疾致病寄生虫约氏疟原虫的內源基因Pyp28分别通过2A和60Linker两种短肽融合表达mCherry红色荧光蛋白。参考PlasmoDB数据库的Pyp28基因序列设计sgRNA、同源重组序列,分别将其连接至pYC载体上。质粒与疟原虫共转染至小鼠体内,5-6天后检测修饰效率,并通过有限稀释法获得单克隆。盐酸苯肼诱导配子体后,饲喂雌性按蚊,并取感染小鼠血液体外培养动合子,观察雌配子/合子、早期动合子、合子、早期卵囊四个阶段mCherry荧光信号,验证P28蛋白的表达。该研究获得了Pyp28內源基因融合mCherry红色荧光蛋白的荧光报告虫株,为研究p28基因及其蛋白质产物的亚细胞定位和相关蛋白质相互作用提供了有效的模型。
关键词: CRISPR/Cas9 P28蛋白 mCherry 荧光报告虫株
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Construction of fluorescent reporter strain by CRISPR/Cas9 in Plasmodiumyoelii sexual stage
Abstract:In this study, we applied the CRISPRC/Cas9-bsed mehthodsto tag the gene p28 with 2A and 60-base pairs-linker peptide mediated mCherry respectively. In according to the sequences of p28 downloaded from the PlasmoDB database, we design the target sites for sgRNA and select suquences as primers for homologous arms. The vector and parasites mixed and transfected, followed by injection into mice. In the presence of pyrimethamine, the parasite genome recombinated with vector through the homologous arms. We got the signle cloning of mCherry tagging strain in limited dilution method. We observe the mCherry signal at zygote, retort ookinete, mature ookinete and parasites in mosquito midgut after 48h infection. through microscope. The p28 tagging with mCherry strain provide a effective model for researching the interact protein.
Keywords: CRISPR/Cas9 P28 protein mCherry Fluorescent reporter strain
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No.4730640119533414****
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利用CRISPR/Cas9构建约氏疟原虫有性阶段的荧光报告虫株
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