Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao, 266071, People's Republic of China.
Fish Physiol Biochem. 2013 Oct;39(5):1239-51. doi: 10.1007/s10695-013-9779-5. Epub 2013 Mar 23.
In this study, the cytochrome P450 3A (CYP3A) gene was cloned from the turbot Scophthalmus maximus for the first time using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends approaches. The amino acid sequences were analyzed with corresponding software programs. The cDNA of CYP3A was 1,969 bp in length, which contained a 5'-untranslated region (UTR) of 34 bp, a 3'-UTR of 404 bp and an open reading frame of 1,530 bp encoding a predicted protein of 509 amino acids (GenBank accession No. JN216889). The deduced protein had a molecular weight of 58.09 kDa and an isoelectric point of 5.75. Amino acid sequence alignment indicated that turbot CYP3A shared 60-67% homology with other fish species. It consists of a signal peptide, six conservative substrate recognition sites (SRS 1-6) and the conserved heme-binding motif FXXGXXXCXG in all CYP3As. Quantitative real-time RT-PCR analysis indicated that turbot CYP3A mRNA was widely expressed in liver, kidney, gill, muscle, stomach, intestine, gallbladder and spleen, with the highest level in liver and the lowest in muscle. After oral administration of sulfamethazine, CYP3A expression in all experimental groups enhanced compared with control, and the expression varied with administration time. It suggested that CYP3A expression could be induced by sulfamethazine. Our findings provided molecular characterization and expression profile of turbot CYP3A, and revealed the important role that turbot CYP3A played in drug metabolisms.
本研究首次采用反转录聚合酶链式反应（RT-PCR）和快速扩增 cDNA 末端方法从大菱鲆 Scophthalmus maximus 中克隆细胞色素 P450 3A（CYP3A）基因。用相应的软件程序对氨基酸序列进行分析。CYP3A 的 cDNA 长 1969bp，包含 34bp 的 5′非翻译区（UTR）、404bp 的 3′UTR 和 1530bp 的开放阅读框，编码一个预测的 509 个氨基酸的蛋白质（GenBank 登录号 JN216889）。推导的蛋白质分子量为 58.09kDa，等电点为 5.75。氨基酸序列比对表明，大菱鲆 CYP3A 与其他鱼类种属的同源性为 60-67%。它由一个信号肽、六个保守的底物识别位点（SRS 1-6）和所有 CYP3A 中保守的血红素结合基序 FXXGXXXCXG 组成。实时定量 RT-PCR 分析表明，大菱鲆 CYP3A mRNA 在肝、肾、鳃、肌肉、胃、肠、胆囊和脾中广泛表达，在肝中表达水平最高，在肌肉中表达水平最低。口服磺胺甲噁唑后，所有实验组 CYP3A 的表达均较对照组增强，且表达随给药时间而变化。这表明磺胺甲噁唑可诱导 CYP3A 的表达。本研究为大菱鲆 CYP3A 的分子特征和表达谱提供了依据，揭示了大菱鲆 CYP3A 在药物代谢中的重要作用。
