生物技术通报Issue(11)：89-91,97,4.
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
郭荫广 1陈全 2朱大冕1
作者信息
- 1. 重庆医科大学分子医学与肿瘤研究中心,重庆400016
- 2. 重庆医科大学基础医学院免疫学教研室,重庆400016
- 折叠
摘要
Abstract
It was to construct a eukaryotic 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 gene fragment encoding GPI of CD24 was amplified by RT-PCR using the obtained RNA as template, which was inserted into the pEGFP-Cl plasmid to construct recombinant plasmid pEGFP-Cl -GPI. Then the plasmid was transfected into A549 cells by Lipofectamine 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-Cl plasmid. The enhanced green fluorescence was observed around the cell membrane of A549 cells transfected with pEGFP-Cl-GPI. As the pEGFP-Cl control cells,the green fluorescence was observed in the cytoplasm uniformly. Therefore,the eukaryotic expression plasmid pEGFP-Cl-GPI was constructed successfully and could express anchored EGFP around the cell membrane, which provides a basis for the construction of anchored tumor vaccine.关键词
GPI/真核表达载体/A549/绿色荧光蛋白Key words
Glycosyl phosphatidylinositol/Eukaryotic expression vector/AS49/Enhanced green fluorescent protein分类
生物科学引用本文复制引用
郭荫广,陈全,朱大冕..糖基化磷脂酰肌醇锚定型EGFP真核表达质粒的构建及表达[J].生物技术通报,2009,(11):89-91,97,4.
