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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

发表时间:2017-11-15  浏览量:1348  下载量:189
全部作者: 刘聪,张翠,袁晶,崔慧婷
作者单位: 厦门大学生命科学学院
摘 要: 利用实验室在疟原虫中建立的CRISPR/Cas9系统,将疟疾致病寄生虫约氏疟原虫的内源基因Pyp28分别通过2A和60Linker(60L)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 d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,同时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4个阶段mCherry荧光信号,验证P28蛋白的表达。该研究获得了Pyp28内源基因融合mCherry红色荧光蛋白的荧光报告虫株,为研究Pyp28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: 分子生物学;荧光报告虫株;CRISPR/Cas9;P28蛋白;mCherry
Title: Construction of fluorescent reporter strain by CRISPR/Cas9 in Plasmodium yoelii sexual stage
Author: LIU Cong, ZHANG Cui, YUAN Jing, CUI Huiting
Organization: School of Life Sciences, Xiamen University
Abstract: In this study, we apply the CRISPR/Cas9-based method to tag the endogenous gene Pyp28 with two short chain polypeptide [2A and 60-base pairs-linker peptide (60L)] for fusion expression of mCherry red fluorescent protein in Plasmodium yoelii, respectively. According to the gene sequences of Pyp28 downloaded from the PlasmoDB database, the target sites of sgRNA and homologous recombination sequences are designed as primers, which are connected to pYC carrier, respectively. The vector and parasites are mixed and transfected, followed by injection into mice, and then the modified efficiency is detected after 5-6 d. The single cloning of mCherry tagging strain is achieved by limited dilution method. Phenylhydrazine-treated mice are used to obtain blood with high gametocytemia for female mosquito-feeding. Also we collect blood for ookinete culture in vitro and observe the mCherry signal at female gamete/zygote, retort ookinete, mature ookinete and parasites in mosquito midgut after 48 h infection through microscope. The fluorescent reporter strain fused by endogenous gene Pyp28 with mCherry red fluorescent protein is achieved in this study, and an effective model is provided for sub-cellular localization of Pyp28 and its related proteins interaction.
Key words: molecular biology; fluorescent reporter strain; CRISPR/Cas9; P28 protein; mCherry
发表期数: 2017年11月第21期
引用格式: 刘聪,张翠,袁晶,等. 利用CRISPR/Cas9构建约氏疟原虫有性阶段的荧光报告虫株[J]. 中国科技论文在线精品论文,2017,10(21):2339-2346.
 
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