人角蛋白K1cDNA基因克隆及其定点突变  

Clone and site-directed mutagenesis of human keratin-1 cDNA gene

作　　者：孙秀坤[1] 朱学骏[1] 

机构地区：[1]北京大学第一医院皮肤性病科

出　　处：《临床皮肤科杂志》2004年第3期139-141,共3页Journal of Clinical Dermatology

基　　金：北京市自然科学基金资助课题(7012020);科技部重大专项课题(2002BA711A07)

摘　　要：目的既往的研究曾发现一先天性大疱性鱼鳞病样红皮病BCIE患者存在K1E477K突变现进一步探讨该突变的致病意义。方法首先克隆出正常人K1cDNA全长编码序列,并以此pK1cDNA重组质粒为模板,采用PCR介导的重叠延伸诱变法对K1第477位密码子GAG→AAG进行定点突变。结果克隆的正常人K1cDNA经测序证实长2022bp,与美国国立生物技术信息中心(NCBI)上所公布序列一致定点突变准确未改变其他序列。结论通过该研究得到了正常人角蛋白K1的基因克隆pK1cDNA及其突变体pMK1cDNA,为以后研究致病基因功能奠定了基础。Objective: Bullous congenital ichthyosiform erythroderma (BCIE) is due to the mutation on keratin-1/10 (K1/10) gene. Our previous study reported a case of BCIE with the mutation on K1E477K [AAG (E) -> AAG (K)]. The study was designed to make the same E477K mutation in order to get insight into the effect of the mutation on K1 gene. Methods: a full-length K1 cDNA open reading frame was cloned. Taking the pK1cDNA as a model, a site-directed mutagenesis of K1E477K was performed by overlap extension using PCR. Results: Sequencing proved that the sequence of cloned full-length K1cDNA was 2022 bp, which was accordant with the sequence in Genebank. The site-directed mutagenesis was accurate, without any other substitution. Conclusion: Through this study, a normal K1 cDNA gene (pK1cDNA) and mutated one (pMK1cDNA) were cloned successfully. It is a good beginning for further study on the K1 gene.

关 键 词：红皮病 大疱性鱼鳞病样 先天性 角蛋白1 基因克隆 定点突变 

分 类 号：R758.67[医药卫生—皮肤病学与性病学]