糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达

Construction of Eukaryotic Expression Plasmid of GPI-anchored EGFP and Its Expression in A549 Cells

构建与增强型绿色荧光蛋白基因相连的糖基化磷脂酰肌醇(glycosyl phosphatidylinositol,GPI)序列的真核表达质粒,并检测其在A549细胞中的表达。分离人外周血淋巴细胞,提取总RNA,以RT-PCR法扩增CD24基因的243bpGPI锚定序列,双酶切后定向克隆入pEGFP-C1质粒中,构建并鉴定pEGFP-C1-GPI质粒。经脂质体介导转染A549细胞后,在荧光显微镜下观察目的蛋白在真核细胞内的表达情况。经酶切和测序鉴定证实,所克隆的CD24GPI序列正确,荧光显微镜观察pEGFP-C1-GPI质粒转染A549细胞可见围绕细胞膜的强绿色荧光,而对照pEGFP-C1质粒转染A549细胞仅见胞内均匀荧光。成功构建与EGFP相连的GPI真核表达质粒,且能在A549细胞膜上锚定表达EGFP-GPI融合蛋白,为构建锚定表达型肿瘤疫苗奠定基础。

It was to construct a eukaryotie expression plasmid of CD24 GPI gene linked with EGFP gene at its N terminal,and observe its expression in A549 cells. Lymphocytes were isolated from human peripheral blood and total RNA was extracted. The gone fragment eneoding GPI of CD24 was amplified by RT-PCR using the obtained RNA as template, which was inserted into the pEGFP-C1 plasmid to construct recombinant plasmid pEGFP-C1-GPI. Then the plasmid was transfeeted into A549 cells by Lipofectaminc 2000, and its expression function was detected by observing the expression of enhanced green fluorescent protein（EGFP）through fluorescent microscope. The results of restriction endonuclease analysis and DNA sequencing proved that the GPI gene fragment was correctly inserted into pEGFP-C1 plasmid. The enhanced green fluoreseence was observed around the cell membrane of A549 cells transfected with pEGFP-C1-GPI. As the pEGFP-C1 control cells,the green fluorescence was observed in the cytoplasm uniformly. Therefore, the eukaryotic expression plasmid pEGFP-C1-GPI was construeted successfully and could express anchored EGFP around the cell membrane, which provides a basis for the construction of anchored tumor vaccine.