绿荧光蛋白的荧光强度在D-氨基酸氧化酶活性检测中的应用  

Application of fluorescence intensity of green fluorescence protein in measuring activity of R.gracilis D-amino acid oxidase

作　　者：翟勇平[1] 王健民[2] 张雨生[2] 周虹[2] 吕书晴[2] 

机构地区：[1]南京军区南京总医院血液科,江苏南京210002 [2]第二军医大学长海医院血液科,上海200433

出　　处：《医学研究生学报》2002年第2期95-97,共3页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目 (批准号 39870 710 )

摘　　要：目的 :探讨在转基因细胞KDfGc和KDfGd,以内部核糖体进入位点 (IRES)序列连接的绿荧光蛋白 (GFP)的荧光强度为单位 ,衡量D 氨基酸氧化酶 (DAAO)活性的准确性。　方法 :白血病细胞系K5 62e经转染含IRES序列连接的GFP和DAAO基因的逆转录病毒载体pLDfG ,获得稳定表达GFP和DAAO基因的单克隆细胞KDfGc和KDfGd,以流式细胞仪和Lowry测定其荧光阳性率以及平均荧光强度、蛋白量 ,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 :KDfGc和KDfGd细胞的荧光阳性率分别为 94 .64%和 96.3 1%。 2× 10 4细胞的平均荧光强度分别为 2 0 2U和 174U。 2× 10 4细胞的蛋白量分别为 13 .17μg和 7.12 μg ,KDfGc、KDfGd细胞每微克蛋白量每小时产生的H2 O2 峰值无差异 ,而以单位荧光来衡量 ,两细胞具有明显差别。　结论 :在含IRES序列的转基因细胞 ,以GFP的平均荧光强度为度量单位 ,比以细胞蛋白量能更准确地反映DAAO的活性。Objectives:To observe the accuracy of fluorescence intensity of green fluorescence protein(GFP) in assessing activity of R.gracilis D amino acid oxidase(DAAO). Methods: K DfGc ,K DfGd cell lines stably expressing DAAO and GFP were obtained by retrovirus transfection which made with vector pLDfG containing IRES sequence,DAAO cDNA and GFP gene. Fluorescence positive rate and mean fluorescence intensity of the two cell lines were measured with flow cytometer. Lowry method was used to assay the protein of cells. H 2 O 2 production by K DfGc ,K DfGd cells were measured by the phenol red oxidation assay. Results:Fluorescence positive rate and mean fluorescence intensity of K DfGc ,K DfGd were 94.64 %,96.31% and 202 U,174 U(2×10 4 cells)respectively. The protein of 2×10 4 K DfGc cells was 13.17 μg, the K DfGd cells's was 7.12 μg. The peaks of H 2 O 2 per microgram protein of K DfGc and K DfGd treating with D Ala for 1 hour were similar, but it was different if assayed by mean fluorescence intensity. Conclusions: In transgeneic cells containing IRES sequence, the activity of DAAO assessed by mean fluorescence intensity of GFP is more accurate.

关 键 词：D-氨基酸氧化酶 绿荧光蛋白 基因表达 活性 检测 应用 

分 类 号：R446.61[医药卫生—诊断学]