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

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

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RT-PCR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。

Objective The purpose of this study was to clone and analyze mutation in the eda-A1 gene for hypohidrotie 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 trancription polymerase chain reaction （RT-PCR） with a pair of specific primers containing the constriction enzyme sites of BamH I and HindⅢ. When the vector pcDNA3.1 （-） and eda-A1 （M/W） were digested by BamH I 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 missenee 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.