绿色荧光蛋白表达载体pXS75-GFP的构建及在嗜热四膜虫中的应用

Construction of Green Fluorescent Protein Expression Vector pXS75-GFP and Application in Tetrahymena thermophila

为获得能够用于构建嗜热四膜虫蛋白定位的载体,该研究将GFP基因与镉(Cd2+)诱导的四膜虫金属硫蛋白基因(MTT1)启动子序列和终止子序列融合,获得表达载体pXS75-GFP。通过同源重组和抗性筛选,pXS75-GFP载体携带的目的基因整合入四膜虫MTT1位点,在Cd2+诱导下实现GFP融合蛋白的可控表达。将α-tubulin基因ATU1克隆入pXS75-GFP中,重组质粒pXS75-GFP-ATU1通过基因枪转化入四膜虫细胞,在巴龙霉素筛选下获得稳定的α-tubulin-GFP过表达细胞株。激光共聚焦显微镜观察α-tubulin-GFP的定位,结果显示,α-tubulin-GFP融合蛋白在四膜虫细胞中表达并分布于皮层上,表明pXS75-GFP载体可用于嗜热四膜虫功能蛋白的定位分析。

To construct a vector for studying the localization of protein in Tetrahymena thermophila, GFP expression vector pXS75-GFP was constructed by ligating GFP with Cd2＋inducible metallothionein （MTT1） promoter and terminator sequences. The target gene-GFP fusion gene can integrate into the MTT1 locus through homologous recombination and resistance screening. Expression of the target protein in-fusion with the α-terminal GFP tag was controllable by Cd2＋. The recombinant plasmid pXS75-GFP-ATU1 was constructed and biolistically transformed into Tetrahymena. The expression of α-tubulin-GFP was analyzed by Western blot. Confocal microscopy showed that α-tubulin-GFP localized at cortex in living and fixed Tetrahymena cells. The results revealed that pXS75-GFP can be used for studying the subcellular localization of proteins in Tetrahymena thermophila.