Yoon K, Thiede M A, Rodan G A
Department of Bone Biology and Osteoporosis Research, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Gene. 1988 Jun 15;66(1):11-7. doi: 10.1016/0378-1119(88)90220-x.
This study examines the use of alkaline phosphatase (AP) as a reporter enzyme. We constructed a plasmid containing the cDNA which encodes the bone/liver/kidney rat AP under the control of the simian virus 40 (SV40) early promoter and used it to transfect Chinese hamster ovary, SV40-transformed African Green Monkey kidney 7, and rat osteosarcoma 25/1 mammalian cells. AP activity in these cells, measured three days later, was 40-400-fold above background. When AP and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected, the detection of AP activity by a simple spectrophotometric assay was at least as sensitive as the detection of CAT activity using a radioactive substrate. Moreover, since mammalian AP is a membrane-bound ectoenzyme, transfected cells can be visualized by histochemical staining. This approach was used to estimate transfection efficiency. The convenient methods for AP detection should make it a useful reporter enzyme.
本研究考察了碱性磷酸酶（AP）作为报告酶的应用。我们构建了一个质粒，其中包含在猴病毒40（SV40）早期启动子控制下编码大鼠骨/肝/肾碱性磷酸酶的cDNA，并使用该质粒转染中国仓鼠卵巢细胞、SV40转化的非洲绿猴肾细胞7以及大鼠骨肉瘤25/1哺乳动物细胞。三天后测量这些细胞中的AP活性，其比背景值高40至400倍。当AP质粒和氯霉素乙酰转移酶（CAT）质粒共转染时，通过简单的分光光度法检测AP活性至少与使用放射性底物检测CAT活性一样灵敏。此外，由于哺乳动物AP是一种膜结合的胞外酶，转染细胞可通过组织化学染色进行可视化。该方法用于评估转染效率。AP检测的便捷方法应使其成为一种有用的报告酶。
