野生型及突变型α-synuclein转基因表达载体的构建

Construction of Transgenic Expression Vector of Wild Type and Mutant Human α-Synuclein

目的构建中枢神经系统特异表达的野生型及突变型-αsynuclein转基因表达载体。方法通过PCR方法扩增-αsynuclein基因,产物亚克隆到pGEM-T载体,经测序无误后克隆到pCEP4载体,并用神经特异的PDGF启动子替代pCEP4载体的CMV启动子构建野生型转基因表达载体。利用大引物突变法构建A53T和A30P的转基因表达载体。结果通过限制性内切酶及DNA测序证实野生型及突变型-αsynuclein均成功的克隆到所需要的载体中。结论该转基因表达载体的成功构建为帕金森氏病转基因动物的构建及帕金森氏病发病机制的研究奠定基础。

Objective To construct the transgenie expression vector of wild type and mutant human α-synuelein which can be specifically expressed in the central nervous system. Methods The full length wild type α-synuelein was amplified by PCR and the PCR product was subcloned into pGEM-T vector. After DNA sequencing,the fragment was cloned into pCEP4 vector,and the CMV promoter was replaced by PDGF promoter. The site-directed mutagenesis by megaprimer PCR method was used to construct the A53T and A30P transgenie expression vector. Results The wild type and mutant α-synuelein was successfully cloned into the needed vector by enzymatic digestion and DNA sequencing. Conclusion This work has laid foundations for the construction of Parkinson＇ s disease（PD）transgenie animal models and for further studies on the role of α-synuelein in Parkinson＇ s disease.