荧光定量检测细胞绿色荧光蛋白技术的建立与应用

Method for Fluorescent Quantification of Cellular Green Fluorescence Protein and Its Application

绿色荧光蛋白是在生化与分子生物学研究领域中普遍使用的一种基因表达报告系统显色物.对于细胞内绿色荧光蛋白表达水平的定量检测,目前尚缺乏一套准确?简便?易行的技术.本文以增强型绿色荧光蛋白(EGFP)为报告基因,用分子量为25 ku的线性聚乙烯亚胺介导进入HeLa细胞并表达.利用多功能酶标仪测定细胞裂解液的荧光强度,结果显示:这种新方法测定的荧光强度能很好反映细胞中EGFP的蛋白表达水平,并在短时间内完成对EGFP表达水平的定量检测.该方法简单有效,且不依赖于大型仪器设备,具有较高的准确性与灵敏度,在实际应用中简便可靠,具有推广应用的意义.

Green Fluorescence Protein（ GFP）, a material of reporter gene expression system, was widely utilized in the different fields of biological research. However, it is difficult to quantitatively measure the GFP level of cells in a simple and effective way. In this study, we set up an easy, sensitive and efficient method. Enhanced Green Fluorescence Protein （EGFP） was transfected into HeLa cells by 25 ku linear polyethylenimine. After preparation of cell lysate, the fluorescence intensity of expressed EGFP can be detected by multifunctional microplate reader without the utilization of expensive apparatus. Our results indicated that the new method is highly stable and precise in application and allows sensitive detection of protein level in HeLa cells. It has been well proved that the fluorescence intensity of EGFP is consistent with the protein level in cells.