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

作　　者：李琳[1] 倪培华[2] 刘振国[1] 陈生弟[1] 

机构地区：[1]上海第二医科大学附属瑞金医院神经内科,帕金森病诊疗与研究中心,200025 [2]上海第二医科大学附属瑞金医院检验系,200025

出　　处：《上海医学》2005年第1期58-60,共3页Shanghai Medical Journal

基　　金：上海市科委青年科技"启明星后"计划(01QMH1410);国家重点基础研究计划"脑功能和脑重大疾病的基础研究"(G1999054008)资助项目

摘　　要：　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.

关 键 词：Α-SYNUCLEIN 突变型 野生型 真核表达质粒 PCDNA3 脑组织 致病机制 酶切 碱基 克隆 

分 类 号：R346[医药卫生—基础医学]