CYP2B7cDNA的克隆,鉴定及真核细胞重组表达载体的构建

CLONING AND IDENTIFICATION OF HUMAN CYTOCHROMECYP2B7 GENE cDNA AND CONSTRUCTION OF ITS MAN-MALIAN CELLE XPRESSION RECOMBINANT

应用ＲＴ—ＰＣＫ技术和ＤＮＡ重组技术从人肝组织中克隆细胞色素Ｐ４５０２Ｂ７（ＣＹＰ２Ｂ７）基因的ＣＤ－ＮＡ片段至ｐＧＥＭ—３Ｚ载体上，经限制性内切酶切图谱分析和ＤＮＡ序列分析确证克隆的片段为ＣＹＰ２Ｂ７的ｃＤＮＡ。然后将该片段亚克隆入一合适真核细胞表达载体的ＢａｍＨＩ位点处，获得４个克隆。其中１个含有正向插入片段，３个含有反向插入片段。为导入哺乳类细胞中，建立稳定表达ＣＹＰ２Ｂ７ｃＤＮＡ的细胞株打下基础。

With reverse transcription polymerase chain reaction(RT PCR)and DNA re-combinant technique ,a full length cDNA encoding the monooxygenase cytochrome P450 ⅡB7 isolated from human liver was cloned into a plasmid pGEM 3Z. The cDNA segment wasanalysed by its restriction map and DNA sequencing,and showed nothing other than CYP287cDNA. Then the CYP2B7 cDNA was subcloned into a mammalian episomal expression vec-tor. Four clones with CYP2B7 cDNA insert were identified,one in right ortentation and threein reverse orientation.