外源性p38调节活化蛋白激酶细胞内定位研究

目的 研究外源性调节活化蛋白激酶 (PRAK)在细胞内的定位.方法 将克隆在pET-14b上的PRAK亚克隆到绿色荧光蛋白载体pEGFP-C2上,随后转染Hela细胞,并通过荧光显微镜观察外源性PRAK导入真核细胞后在细胞内的分布.结果 重组质粒经酶切、PCR和测序鉴定正确无误,并在Hela细胞中得到高量表达.融合蛋白发出的绿色荧光表明EGFP-PRAK主要分布在细胞核中.结论 成功构建了PRAK绿色荧光蛋白融合载体,该载体能在哺乳动物细胞中进行表达,通过荧光显微镜观察外源性PRAK主要分布在细胞核内.

Research on Localization of Exogenous p38 Regulated/Activated Protein Kinase (PRAK) in Cells

Objective To study the localization of p38 regulated/activated protein kinase (PRAK) in the cells. Methods PRAK with His-tag in pET-14b was subcloned into the green fluorescent protein vector, pEGFP-C2. The construct was then transfected into Hela cells, which were observed by fluorescent microscope later. Results After identification by enzyme digestion, PCR and sequencing, the recombinant vector was highly expressed in Hela cells. The green fluorescence of fusion protein showed that EGFP-PRAK molecules were mainly in the cellular nuclei. Conclusions A vector of PRAK fused with green fluorescent protein is successfully constructed and can express in mammalian cells. The exogenous PRAK molecules are mainly observed in the cellular nuclei.