IRES序列连接的D-氨基酸氧化酶与绿色荧光蛋白基因导入K562e细胞的表达

内部核糖体进入位点 (IRES)序列来源于脑心肌炎病毒 ,它可翻译一条mRNA上的两个开放读框 ,由其连接的两个基因的表达率相同。本实验应用以IRES序列连接D 氨基酸氧化酶 (DAAO)和绿色荧光 (GFP)基因构建的逆转录病毒载体 ,转染K5 6 2e细胞获得稳定表达GFP的单克隆细胞KDfGC和KDfGd,测定转基因细胞的荧光阳性率以及荧光强度 ,用D 丙氨酸 (D Ala)作用DAAO+细胞 ,并用酚红氧化法测定培养上清H2 O2 。结果表明 ,KDfGC和KDfGd细胞的荧光阳性率分别为 94 .6 4 % ,96 .31% ;2× 10 4细胞的荧光强度分别为 2 0 2U和 174U。GFP荧光强度与H2 O2 的产量间呈指数相关。结论 :IRES序列调控的双基因可同时表达 ,GFP的荧光强度可以间接地反映DAAO基因的表达水平 ,为了解目的基因表达水平高低提供了新的方法。

Expression of D-Amino Acid Oxidase Gene and Green Fluorescence Protein Gene Transferred into K562e Cells by Retroviral Vector Containing Internal Ribosome Entry Site Sequence

The internal ribosome entry site (IRES) sequence was derived from encephalomyocarditis virus. It allows to translate two open reading frames at one mRNA, so two genes conjoined by IRES have the same expression rate. K(DfGC) and K(DfGd) cell lines, stably expressing D-amino acid oxidase (DAAO) gene and green fluorescence protein (GFP) genes, were obtained by transfection of K562e cells with retroviral vector pLDfG containing IRES sequence, DAAO cDNA and GFP gene. Fluorescence positive rate and fluorescence intensity of the two cell lines were measured with flow cytometry. H(2)O(2) production by K(DfGC) and K(DfGd) cells treated with D-alanine was measured by the phenol red oxidation assay. The fluorescence positive rate and fluorescence intensity in K(DfGC) and K(DfGd) cell were 94.64% and 96.31% and 202 units and 174 units per 2 x 10(4) cells, respectively. There was exponential correlation between fluorescence intensity and H(2)O(2) level. The above-mentioned results demonstrate that DAAO gene and GFP gene were simultaneously expressed in K562e cell line by the regulation of IRES sequence, and DAAO level was correlated with fluorescence intensity of GFP.