野生型和G88C突变型α-synuclein基因真核表达质粒构建

Construction of eukaryotic expression system of wild type and G88C mutant human α-synuclein

目的 构建人野生型和突变型 (G88C)α- synuclein基因真核表达载体。方法 采用逆转录 聚合酶链反应从人胚脑组织中扩增人α -synuclein基因,克隆至pMD18 T中。采用SOE法定点突变获得G88C突变型α- synuclein基因,并将其克隆至pMD18 T中。酶切鉴定后将野生型和突变型 (G88C)α- synuclein基因亚克隆至真核表达质粒pcDNA3. 1+中。结果 酶切及DNA测序结果证实,野生型和突变型α- synuclein基因分别插入到pcDNA3. 1+中,野生型α- synuclein基因序列与基因库 (登录号L08850)完全一致,突变型 (G88C)α- synuclein基因除第 88位碱基G被C替代以外,其余序列与野生型完全一致。结论 构建野生型和G88C突变型α- synuclein基因真核表达质粒为研究人α- synuclein基因的功能及其突变体的致病机制奠定了基础。

Objective To construct the expression system of human wild type and mutant G88C α-synuclein. Methods The cDNA encoding human α-synuclein was isolated by using RT-PCR method with total RNA extracted from fetal brain. The mutant G88C α-synuclein was obtained by gene splicing and overlap extension (SOE) on site-directed mutagenesis of wild-type human α-synuclein. The wild type and mutant G88C α-synuclein genes were cloned into pcDNA3.1+ and identified by restriction enzyme analysis and sequencing. Results The wild type and mutant α-synuclein genes were successfully cloned into pcDNA3.1+. The sequence of the wild type α-synuclein showed the same sequence as that in the Gene bank, and the sequence of mutant α-synuclein was concordant with that of the wild type α-synuclein except that the number 88 base G was replaced by C. Conclusion The construction of an expression system of wild type and mutant G88C α-synuclein lays down a foundation for investigating the pathogenesis of Parkinson's disease.