人细胞色素p450IA1基因cDNA的克隆和鉴定

Cloning and identification of human cytochrome p450ⅠA1 gene cDNA

在用３－甲基胆蒽诱导培养人羊膜ＦＬ细胞２４ｈ后，抽提细胞总ＲＮＡ并直接合成ｃＤＮＡ第一链。利用人工合成的一对寡核苷酸引物，采用ＰＣＲ技术特异性地扩增Ｃｙｔｐ４５０ⅠＡ１ｃＤＮＡ。３０个循环后琼脂糖凝胶电泳显示１．５Ｋｂ大小片段，长度与预计相符。Ｓｏｕｔｈｅｒｎ杂交结果证实此片段确为Ｃｙｔｐ４５０ⅠＡ１ｃＤＮＡ。将此片段克隆至质粒ｐＧＥＭ－３Ｚ并进行部份序列分析。结果显示克隆片段包含Ｃｙｔｐ４５０ⅠＡ１ｃＤＮＡ５＇端和３＇端部份非编码区及完整的编码区。

Total RNA was isolated from the FL cells which was induced with 3-methylcholanthrene for 24 hours.The first strand of cDNA was synthesized from itusing reverse transcriptase.Polymerase chain reaction (PCR) was used to amplify Cytp450ⅠA1 cDNA with two synthetic oligonucleotide primers in which BamHI and XhoIcleavate sites were engineered in the 5'and 3'primers respectively. After PCR amplifi-cation of 3 cycles,a sharp DNA band of 1.5 Kb size can be visualized in the expec-ted position after electrophoretic separation.Southern blot analysis showed that it is apositive band. The amplified human Cyt p450ⅠA1 fragment was purified and recoveredon a 1.5% agarose gel, followed by cloning intothe polycloning site of vector pGEM-3Z.Partial sequencing data revealed that the cloned segment consist of 1570 bp, inclu-ding 5'-and 3'-untranslated region and a complete coding framework of that gene.