CYP1A1基因cDNA全长的T载体克隆及序列分析  

The full-length cDNA of CYP1A1 gene cloning by T vector and sequencing

作　　者：肖勇梅[1] 刘移民[2] 魏青[1] 招小林[1] 侯孟君[1] 凌文华[1] 

机构地区：[1]中山大学公共卫生学院预防医学系,广州510080 [2]广州市职业病防治院

出　　处：《毒理学杂志》2006年第1期7-9,共3页Journal of Toxicology

基　　金：广东省自然科学基金(015042);中华医学基金会(CMB)资助

摘　　要：目的构建含KpnI、XhoI双酶切位点的人细胞色素(CYP1A1)基因cDNA全长的pGEM-T载体,为以后的亚克隆和毒理学研究提供实验材料。方法从培养的人乳腺癌细胞(MCF-7)中抽提总RNA,RT-PCR扩增CYP1A1基因cDNA全长,与pGEM-T载体连接后转化DH5α,筛选阳性克隆,抽提重组质粒并进行PCR及酶切鉴定,再行测序分析。结果重组质粒pGEM-T-1A1 PCR后获得了1 568 bp产物,酶切鉴定证实目的片段成功插入至pGEM-T,测序分析也进一步证明了目的片段与GeneBank中CYP1A1mRNA的序列同源性为99.9%。结论成功地构建了含CYP1A1基因cDNA全长区域的T载体。Objective To construct the pGEM-T vector containing the whole cDNA of human cytochrome 1A1（CYP1A1）gene with two recognition sites for the enzymes KpnI and XhoI and provide cxperimental, material for subclone and toxicological study. Methods The total RNA from cultured MCF-7 cells was extracted and the full-length cDNA of CYP1A1 mRNA was cloned by RT-PCR. The amplified DNA fragments were ligated into pGEM-T vector, and then transformed into E. coil strain DHSa. The positive clones were screened out and recombinant plasmids were isolated from such colonies. The recombinants were idenified by PCR, restriction endonucleases analysis and sequencing. Results PCR assay showed that a DNA fragment of 1 568 bp could be amplified from the recombinant plasmids pGEM-T-1A1. Restriction endonucleases（ KpnI and XhoI）digestion confirmed the target fragment had been inserted into pGEM-T successfully. The sequencing results verified that the target fragment had 99.9% homology with the CYP1A1 mRNA sequence in Genebank. Conclusion The T-vector clone with the whole cDNA of cytoehrome IAI gene has been constructed successfully.

关 键 词：细胞色素P450(CYP1A1) T-载体 测序分析 

分 类 号：R994.6[医药卫生—毒理学]