野生型和C279G突变型UCHL1基因真核表达质粒构建

Construction of Expression System of Wild and C279G Mutant UCHL1

目的构建人野生型和C279G突变型pEGFP-N1-UCHL1质粒。方法采用RT-PCR方法从人胚脑组织中扩增出人泛素羧基末端水解酶1(UCHL1)基因,插入至pMD18Tvector中;再采用SOE法定点突变,通过酶切和连接,构建野生型和C279G突变型pEGFP-N1UCHL1。结果酶切和DNA测序证实,野生型和突变型UCHL1基因分别插入到pEGFP-N1中,野生型UCHL1基因序列与GenBank完全一致,C279G突变型UCHL1基因除第279位碱基C被G替代以外,其余序列与野生型完全一致。结论成功构建野生型和C279G突变型UCHL1基因真核表达质粒。

Objective To construct the human wild type and C279G mutant pEGFP-N1-UCHL1 plasmids. Methods The cDNA encoding the wild type ubiquitin carboxyl-terminal hydroxylase L1(UCHL1)was isolated by using RT-PCR method from the human fetal brain. C279G mutant UCHL1 was obtained through gene splicing by overlap extension (SOE) on site direct mutagenesis of wild-type human UCHL1. The wild type and C279G mutant UCHL1 gene were cloned into pEGFP-N1, and identified by restriction enzyme analysis and sequencing. Results The wild type and mutant UCHL1 gene were successfully cloned into pEGFP-N1.The sequence of the wild type UCHL1 showed the same to that in the GenBank, and the sequence of mutant UCHL1 was in accordance with that of wild type UCHL1 except that the number 279 base C was replaced by G. Conclusion The construction of an expression system of the wild type and C279G mutant UCHL1 establishes the foundation for the research on the role of UCHL1 in the mechanism of Parkinson’s disease.