Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa -: A novel secreted bioluminescent reporter enzyme
Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa -: A novel secreted bioluminescent reporter enzyme
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DOI：
10.1074/jbc.m309639200
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发表时间：
2004-01-30
影响因子：
4.8
通讯作者：
Vysotski, ES
中科院分区：
文献类型：
--
作者：
Markova, SV;Golz, S;Vysotski, ES
长腹水蚤（Metridia longa）是一种海洋桡足类动物，其表皮腺体会因各种刺激而分泌一种物质，从而产生蓝色生物发光现象。我们证明长腹水蚤荧光素酶对腔肠素具有特异性，可产生蓝光（最大波长=480纳米）。通过一个表达型cDNA文库和功能性筛选，我们克隆并测定了编码长腹水蚤荧光素酶的cDNA序列。该cDNA是一个897碱基对的片段，具有一个656碱基对的开放阅读框，它编码一个含219个氨基酸的多肽，分子量为23885。该多肽包含一个由17个氨基酸残基组成的用于分泌的N端信号肽。在哺乳动物中国仓鼠卵巢细胞中表达长腹水蚤荧光素酶基因时，在培养基中检测到了荧光素酶，这证实了在克隆的荧光素酶中存在一个天然的用于分泌的信号肽。这种新型分泌型荧光素酶在一个实际的检测应用中进行了测试，在该应用中检测到了A2a和NPY2 G蛋白偶联受体的活性。这些结果清楚地表明，分泌型长腹水蚤荧光素酶非常适合作为一种报告基因用于监测基因表达，特别是用于开发新型超高通量筛选技术。
Metridia longa is a marine copepod from which a blue bioluminescence originates as a secretion from epidermal glands in response to various stimuli. We demonstrate that Metridia luciferase is specific for coelenterazine to produce blue light (lambda(max)=480 nm). Using an expression cDNA library and functional screening, we cloned and sequenced the cDNA encoding the Metridia luciferase. The cDNA is an 897-bp fragment with a 656-bp open reading frame, which encodes a 219-amino acid polypeptide with a molecular weight of 23,885. The polypeptide contains an N-terminal signal peptide of 17 amino acid residues for secretion. On expression of the Metridia luciferase gene in mammalian Chinese hamster ovary cells the luciferase is detected in the culture medium confirming the existence of a naturally occurring signal peptide for secretion in the cloned luciferase. The novel secreted luciferase was tested in a practical assay application in which the activity of A2a and NPY2 G-protein-coupled receptors was detected. These results clearly suggest that the secreted Metridia luciferase is well suited as a reporter for monitoring gene expression and, in particular, for the development of novel ultra-high throughput screening technologies.