Construction of promoter-detection plasmid in animal cells by using the metapyrocatechase gene

为了检测中庸儿茶酶（C230）基因在动物细胞中的表达能力，我们首先构建了两个质粒。在一个质粒中，C230基因受SV40早期基因的启动子和增强子控制，而在另一个质粒中，C230基因受人伸长因子基因的启动子和增强子控制。这些质粒在HeLa和CHO细胞中均未显示活性C230的产生。接下来，我们将鸡腺苷酸激酶的n端两个残基连接到n端截断的C230上进行基因融合。将上述两个质粒中的C230基因进行基因融合置换，并检测质粒产物在动物细胞中的表达情况。然而，结果是消极的。由于这些质粒的转录被认为在动物细胞中是活跃的，因此C230基因的表达失败可能是由于翻译后的过程中存在障碍。在本研究过程中，我们获得了一个质粒pKS230，其中C230基因受到lac启动子的控制。HincII和EcoRV的限制性内切位点可以作为外源DNA的克隆位点，因为插入这些位点会使质粒失去活性，从而产生活性C230。在喷洒儿茶酚溶液后，插入的质粒很容易被黄色显色检测到。因此，与使用X-Gal作为检测器的lac启动子载体相比，该质粒可作为成本较低的克隆载体。

To test the ability of expression of the metapyrocatechase (C230) gene in the animal cells, we first constructed two plasmids. In one plasmid, the C230 gene was put under the control of the promoter and enhancer of SV40 early genes, while in the other, it was controlled by the promoter and enhancer of the human elongation factor gene. Neither of these plasmid showed production of active C230 in HeLa and CHO cells. Next, we made a gene fusion in which the N-terminal two residues of chicken adenylate kinase was ligated to the N-terminal truncated C230. The C230 gene of the above two plasmids was replaced by the gene fusion, and the plasmid products were checked for their expression in the animal cells. However, the results were negative. Since the transcription of these plasmids were thought to be active in the animal cells, failure in the expression of the C230 gene is probably due to a barrier in the process after the translation.During the course of this study, we obtained a plasmid pKS230, in which the C230 gene is put under the control of the lac promoter. The restriction sites for HincII and EcoRV can be used as a cloning site for foreign DNA, since insertion to these sites make the plasmid inactive to produce the active C230. The inserted plasmid is easily detected by yellow color development after spraying a catechol solution. Therefore, this plasmid can be used a less expensive cloning vector comparing with the lac promoter vector that uses X-Gal as a detector.