人Ⅰ型醇脱氢酶基因的克隆及其等位基因鉴定

CLONING AND IDENTITY OF THE HUMAN CLASS ⅠALCOHOL DEHYDROGENASE GENE

[目的]克隆人Ⅰ型醇脱氢酶基因,并对所克隆序列进行鉴定。[方法]用RT-PCR法获得Ⅰ型醇脱氢酶基因的cDNA,然后用双酶切得到目的基因并定向克隆到pUC19载体上。经蓝白筛选后,用限制性酶谱和DNA测序以及BLAST比对分析,并结合Internet数据库和相关文献对基因进行鉴定。[结果]证实从一位病人癌旁正常肝组织中克隆的序列有人Ⅰ型醇脱氢酶基因的ADH1B*2、ADH1C*2和ADH1C*2,3个等位基因。[结论]用一对引物成功克隆了人Ⅰ型醇脱氢酶基因的3个等位基因,为进一步研究和利用Ⅰ型醇脱氢酶奠定了基础。

[Objective] To clone the human class I alcohol dehydrogenase gene and identify the sequence. [Methods] The cDNA of the human class Ⅰ alcohol dehydrogenase gene was amphfied by RT-PCR. Then the fragment of the human class I alcohol dehydrogenase gene between two endonuclease sites was obtained and cloned directly into pUC19 vector. After the blue/white screening, the sequence was identified by restriction mapping, DNA sequencing analysis, BLAST alignment and Intemet database and literature searching. [ Results] It was proved that the cloning sequences cloned from normal liver tissue beside of cancer of a patient were ADH1B ＊ 2, ADHIC ＊ 1 and ADHIC ＊ 2 alleles. [Conclusion] The ADH1B ＊ 2, ADH1C ＊ 1 and ADH1C ＊ 2 alleles of the human class Ⅰ alcohol dehydrogenase gene are cloned successfully using a pair of primers, which provides a basis for further research and exploit of the human class Ⅰ alcohol dehydrogenase.