草鱼肾细胞中细胞色素P450 3A基因诱导表达及其酶活性分析

Induction of CYP3A gene and enzyme activity expression in kidney cell of grass carp(Ctenopharyngodon idellus)

采用基因克隆技术获取草鱼(Ctenopharyngodon idellus)细胞色素P450 3A(CYP3A)cDNA序列长1 849 bp,包含完整开放阅读框(open reading frame,ORF)1 542 bp,1个终止密码和含polyA信号3′UTR;ORF编码514个氨基酸,含信号肽(29 aa),2个跨膜螺旋区(23 aa,23 aa),血红素结合域(21 aa)和6个底物识别位点(SRS1-6);与其他脊椎动物CYP3A氨基酸序列相似度达60%～92%。用实时定量PCR和分光光度计法分别研究了草鱼肾细胞系(Ctenopharyngodon idellus kidney cell line CIK)中利福平(rifampicin,RIF)诱导CYP3A基因表达和红霉素-N-脱甲基酶(ERND)活性,采用红霉素脱甲基酶活性法测定CYP3A的活性。结果显示,诱导组CIK细胞中CYP3A mRNA表达量在8 h最高,而CYP3A酶活在10 h最高;CYP3A mRNA和酶活性与对照组均具显著差异性(P<0.05)。CYP3A转录水平先于相应酶活变化表达,且转录水平表达显著高于酶活性,这很可能反映了RIF对CYP3A的诱导主要作用于转录水平而不直接作用于酶活,本研究旨为药物对CYP3A转录调控和活性调节提供科学依据,并为渔药代谢酶的研究提供理论依据。

Cytochrome P450 3A（CYP3A） is the major cytochrome P450 responsible for the metabolism of en-dogenous and xenobiotic substrates.Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity,and can lead to the formation of drug residues,which threaten the safety of fishery products.We determined the in vitro effects of rifampicin（RIF） on CYP3A mRNA expression and enzyme activity in a freshwater teleost,the grass carp（Ctenopharyngodon idellus）.Recently,a number of CYP3A genes have been isolated from several teleost species.Although CYP3A genes in fish contain multiple paralogs differing in gene expression pattern and tissue distribution,CYP3A enzymes exhibit similar catalytic properties because of their structural similarities.The CYP3A gene and its full-length cDNA of 1 849 bp were cloned from grass carp by RT-PCR.The CYP3A gene has an open reading frame（ORF） of 1 542 bp,a stop codon,a 3＇-untranslated regions（3＇ UTR）,and a polyA signal is present in the 3＇UTR region.The ORF encodes a 513 amino acid（aa） protein,which has a signal peptide（29aa）,two transmembrane helixes（23aa and 23aa）,a heme binding domain（HBD,21aa）,and six substrate recognition sites（SRS1-6）.Multiple alignments showed that the CYP3A amino acid se-quence from grass carp is 60% to 92% similar to other vertebrate CYP3As.CYP3A gene expression after induc-tion by RIF in grass carp kidney cells（CIK） was assayed by quantitative real-time PCR.CYP3A-dependent erythromycin N-demethylase（ERND） activity was determined by spectrophotometry,and CYP3A activity was assessed by measuring the formation of formaldehyde.In the induced group,CYP3A mRNA expression reached a plateau at 8h,while the highest level of CYP3A activity occurred at 10h.The results indicated that CYP3A mRNA expression and enzyme activity were significantly higher than in the control group（P0.05）.The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity.In addition,the transcription of level CYP3A was apparently higher than the enzyme activity,implying that induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.