Development of a gene expression vector using Vargula luciferase cDNA.

为了阐明基因在哺乳动物细胞中的表达机制，有必要检测其在各种细胞中的启动子活性。通常情况下，启动子区域与报告基因相连，如大肠杆菌的氯霉素乙酰转移酶(CAT)基因。将杂合基因导入细胞中的CAT活性作为各自基因的启动子活性。最近，利用萤火虫荧光素氧化荧光素而发光的荧光素酶作为报告基因，这种检测方法比普通的CAT检测方法更简单、更灵敏。海洋介形类甲壳动物海尔根多菲是一种具有夜间习性的小动物。当受到干扰时，它会将大量的荧光素和荧光素酶喷射到海水中，产生明亮的发光云。这种光是由荧光素酶催化的酶-底物反应的结果。我们从该动物中纯化了荧光素酶，并测定了其部分氨基酸序列。使用一个集合。在与该氨基酸序列相对应的寡核苷酸中，分离到了Vargula荧光素酶的全长cDNA。该荧光素酶由555个氨基酸组成，含有信号序列。然后将Vargula荧光素酶CDNA置于哺乳动物基因SV40、RSV、延伸因子α(EF-α)和粒细胞集落刺激因子(G-CSF)的启动子下，并导入不同的细胞系。根据其启动子活性，在培养液中检测到不同数量的荧光素酶，表明Vargula荧光素酶是从哺乳动物细胞中高效分泌的。该方法的灵敏度远高于CAT法和萤火虫荧光素酶测定法。我们目前正在尝试建立荧光素酶表达载体稳定整合到染色体中的转化子。使用这种稳定的转化子，就有可能检测活细胞中的基因表达。

To elucidate the gene expression mechanism in mammalian cells, it is necessary to examine its promoter activity in various cells. Usually, the promoter region is connected to the reporter gene such as E. coli chrolamphenicol acetyltransferase (cat) gene. The CAT activity in the cells transfected by the hybrid gene is regarded as the promoter activity of the respective gene. Recently, the firefly luciferase, which emits light by oxidizing luciferin, is used as a reporter gene, and it is shown that this assay method is more simple and sensitive than the ordinary CAT assay.The marine ostracod crustacean Vargula hilgendorfii is a small animal with nocturnal habits. When disturbed, it ejects a copious secretion of luciferin and luciferase into sea water, producing a bright luminous cloud. The light results from an enzyme-substrate reaction, catalyzed by luciferase. We have purified the luciferase from this animal, and its partial amino acid sequence was determined. Using a set. of oligonucleotides corresponding to the amino acid sequence, the full-length CDNA for the Vargula luciferase was isolated. The luciferase is consisting of 555 amino acids, and contains the signal sequence. The Vargula luciferase CDNA was then placed under the promoter of mammalian genes such as SV40, RSV, elongation factor lalpha (EF-lalpha) and granulocyte colony-stimulating factor (G-CSF), and introduced intovarious cell lines. Dependent on its promoter activity, various amounts of luciferase were detected in the medium suggesting Vargula luciferase was efficiently secreted from mammalian cells. The sensitivity of the assay was much higher than that of the CAT assay, and the assay using the firefly luciferase. We are currently trying to establish transformants in which the luciferase expression vector is stably integrated in the chromosomen. Using such stable transformants, it would be possible to examine the gene expression in living cells.