华西口腔医学杂志

• 基础研究 • 上一篇    下一篇

少汗型外胚层发育不良症eda-A1基因突变分析及其真核表达载体的构建

雷科1 车团结2,3 王锦明3 邓旎1 张琳1 何祥一1   

  1. 1.兰州大学口腔医院口腔修复科; 2.兰州大学生命科学院细胞生物学研究所;3.兰州百源基因技术有限公司, 甘肃兰州730000
  • 收稿日期:2009-12-25 修回日期:2009-12-25 出版日期:2009-12-20 发布日期:2009-12-20
  • 通讯作者: 何祥一,Tel:13038730686
  • 作者简介:雷科(1983—),男,四川人,硕士
  • 基金资助:

    甘肃省科技攻关计划资助项目(0709TCYA053)

Mutation analysis of the eda-A1 gene for hypohidrotic ectodermal dysplasia and construction of recombined eukaryotic expression vector

LEI Ke1, CHE Tuan-jie2,3, WANG Jin-ming3, DENG Ni1, ZHANG Lin1, HE Xiang-yi1   

  1. 1. Dept. of Prosthodontics, School of Stomatology, Lanzhou University, Lanzhou 730000, China; 2. Institute of Cell Biology, School of Life Science, Lanzhou University, Lanzhou 730000, China; 3. Baiyuan Genetic Technology Company, Lanzhou 730000, China
  • Received:2009-12-25 Revised:2009-12-25 Online:2009-12-20 Published:2009-12-20
  • Contact: HE Xiang-yi,Tel:13038730686

摘要:

目的克隆少汗型外胚层发育不良症(HED)eda-A1基因并进行突变分析,同时构建eda-A1基因编码序列突变型(M)和野生型(W)的真核表达载体pcDNA3.1(-)-eda-A1-M/W,为进一步明析eda基因的功能奠定基础。方法设计含有BamHⅠ和HindⅢ的酶切位点的引物, 从HED患者和正常人外周血淋巴细胞中提取总mRNA,利用RTPCR法克隆eda-A1(M/W)基因cDNA序列;并运用BamHⅠ和HindⅢ双酶切pcDNA3.1(-)载体和eda-A1基因片段,将eda-A1 (M/W) 插入到pcDNA3.1 (-) 载体中,从而构建新的真核表达载体,命名为pcDNA3.1 (-)-eda-A1-M和pcDNA3.1(-)-eda-A1-W。结果成功克隆少汗型外胚层发育不良症eda-A1基因,并发现未见报道过的907位A→C错义突变,导致306位编码氨基酸由谷氨酰胺变异为脯氨酸;经PCR法、重组载体双酶切法、DNA测序验证,成功构建了pcDNA3.1(-)-eda-A1-W/M的真核表达载体。结论成功构建少汗型外胚层发育不良症eda-A1基因(突变型和野生型)真核表达载体pcDNA3.1(-)-eda-A1-M/W,为进一步研究该基因在牙齿发育中的生物学作用奠定了实验基础。

关键词: 少汗型外胚层发育不良症, eda-A1基因, 突变, 真核表达载体

Abstract:

Objective The purpose of this study was to clone and analyze mutation in the eda -A1 gene for hypohidrotic ectodermal dysplasia(HED), and to construct a new recombined eukaryotic expression vector(mutant M, wild W) as a basis for further study on the genetic function. Methods After total mRNA was extracted from peripheral blood lymphocytes from the HED affect patient and control, eda-A1 gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) with a pair of specific primers containing the constriction enzyme sites of BamHⅠ and HindⅢ. When the vector pcDNA3.1(-) and eda-A1(M/W) were digested by BamHⅠ and HindⅢ respectively, eda-A1(M/W) fragment was then ligated to vector pcDNA3.1(-) and the new vector was named as pcDNA3.1(-)-eda-A1-M/W. Results eda-A1 gene was successfully cloned and a novel missence mutation was identified, which changes the codon 306 from glutamine to proline. PCR, restrictive endonuclease analysis and DNA sequencing were then performed to identify the recombinant eukaryotic expression vector pcDNA3.1(-)-eda-A1-M/W, and the results were surely confirmed. Conclusion Our result indicates that the novel missense mutation in eda is associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level. And also, the recombinant eukaryotic expression vector pcDNA3.1 (-)-eda-A1-M/W was successfully constructed, which will be thereafter taken use of further study on eda gene in odontogenesis.

Key words: hypohidrotic ectodermal dysplasia, eda-A1 gene, mutation, eukaryotic expression vector