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目的 :克隆人细胞色素P45 0 2A6cDNA。方法 :用逆转录 -PCR和DNA重组技术 ,从人肝组织中扩增人细胞色素P45 0 2A6基因的cDNA ,并将其连接到pBluescript载体上 ,对CYP2A6cDNA进行全序列测定。结果 :所克隆的cDNA与Yamano等发表的CYP2A6cDNA序列相比 ,在编码区有两个变异 ,即codon 8CTG→TTG ,编码的氨基酸不变 ,为亮氨酸。codon479GGC(甘氨酸 )→GTC(缬氨酸 ) ,其余均相同。而在其 3′端非编码区变异很多。与Fernandez -Salguero等报道的CYP2A7序列相比 ,其 5′端虽与所克隆的CYP2A6有较多差异 ,但其codon479也是GTC(缬氨酸 ) ,在 3′端非编码区仅略有差异。而所克隆的CYP2A6cDNA与Yamano等报道的CYP2A7序列相比 ,3′端非编码区至所设PCR引物止 ,所克隆的基因序列完全相同 ,而在编码区却差异较多。结论 :本实验室克隆的CYP2A6cDNA是一种新的CYP2A6cDNA序列 ,有可能来自于新的CYP2A6等位基因的转录
Objective: To clone human cytochrome P45 0 2A6 cDNA. METHODS: cDNA of human cytochrome P45 0 2A6 was amplified from human liver tissue by reverse transcription-polymerase chain reaction (PCR) and DNA recombination technique. The cDNA of human P45 0 2A6 gene was amplified and ligated into pBluescript vector. The CYP2A6 cDNA was sequenced. Results: Compared with the CYP2A6 cDNA sequence published by Yamano et al., The cloned cDNA had two mutations in the coding region, namely codon 8CTG → TTG. The encoded amino acid was leucine. codon479GGC (glycine) → GTC (valine), the rest are the same. However, there are many variations in the 3 ’untranslated region. Compared with the CYP2A7 sequence reported by Fernandez-Salguero et al., Its 5 ’end has more differences than the cloned CYP2A6, but its codon479 is also GTC (valine), with only a slight difference in the 3’ non-coding region. However, compared with the CYP2A7 sequence reported by Yamano et al., The cloned CYP2A6 cDNA had the same sequence from the 3 ’non-coding region to the set of PCR primers, but the coding region was quite different. Conclusion: Our laboratory cloned CYP2A6 cDNA is a new CYP2A6 cDNA sequence, which may be derived from the transcription of the new CYP2A6 allele