Qian Jin-jun, Cheng Yan-bo, Liu Chun-feng, Yang Ya-ping, Liu Kang-yong
Department of Neurology, Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Jan;24(1):23-6.
To construct recombinant eukaryotic expression vectors pEGFP-C3-SNCA containing human wild-type (WT) and pathogenic mutations A30P, A53T alpha-synuclein (SNCA), and to obtain monoclonal PC12 cell lines overexpressing human wild-type and pathogenic mutations A30P, A53T alpha-synuclein by stable transfection.
Human wild type SNCA gene was cloned by using RT-PCR. By T-A extension cloning, the gene was ligated with T-vector and sequenced. Based on it, the recombinant eukaryotic expression vectors containing wild type SNCA synonymous mutation or its G88C (Ala30Pro) and G209A (Ala53Thr) pathogenic mutation were constructed by site-directed mutagensis using primer variance in mononucleotide. And different recombinant plasmids pEGFP-C3-SNCA were identified with PCR, restriction enzyme digestion and DNA sequencing. PC12 cells were transfected with different recombinant plasmids pEGFP-C3-SNCA by liposome transfection method, screened with G418, and subcloned by limited dilution method to obtain different monoclonal PC12 cell lines stably over-expressing human wild-type, A30P or A53T alpha-synuclein, respectively, and PC12 cells stably transfected with pEGFP-C3 were used as control group. These monoclonal PC12 cell lines were identified with RT-PCR, Western blot and fluorescence microscopy.
According to result of PCR, the double digestion and gene sequencing, it was comfirmed that recombinant eukaryotic expression vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutation had been constructed successfully. By means of RT-PCR, Western blot and fluorescence microscope, it was comfirmed that objective gene sequences in PC12 cells were stably over-expressed.
The recombinant pEGFP-C3-SNCA vectors containing wild type SNCA synonymous mutation or its Ala30Pro and Ala53Thr pathogenic mutations had been constructed successfully. The transfected monoclonal PC12 cells are obtained in which three SNCA gene isoforms had stable expression.
构建含人野生型（WT）及致病突变A30P、A53Tα-突触核蛋白（SNCA）的重组真核表达载体pEGFP-C3-SNCA，并通过稳定转染获得过表达人野生型及致病突变A30P、A53Tα-突触核蛋白的单克隆PC12细胞系。
采用RT-PCR克隆人野生型SNCA基因。通过T-A延伸克隆将该基因与T载体连接并测序。在此基础上，利用单核苷酸引物变异通过定点诱变构建含野生型SNCA同义突变或其G88C（Ala30Pro）和G209A（Ala53Thr）致病突变的重组真核表达载体。用PCR、酶切和DNA测序鉴定不同的重组质粒pEGFP-C3-SNCA。采用脂质体转染法将不同的重组质粒pEGFP-C3-SNCA转染PC12细胞，用G418筛选，通过有限稀释法亚克隆，分别获得稳定过表达人野生型、A30P或A53Tα-突触核蛋白的不同单克隆PC12细胞系，将稳定转染pEGFP-C3的PC12细胞作为对照组。用RT-PCR、Western印迹和荧光显微镜对这些单克隆PC12细胞系进行鉴定。
根据PCR、双酶切和基因测序结果，证实成功构建了含野生型SNCA同义突变或其Ala30Pro和Ala53Thr致病突变的重组真核表达载体。通过RT-PCR、Western印迹和荧光显微镜证实PC12细胞中目的基因序列稳定过表达。
成功构建了含野生型SNCA同义突变或其Ala30Pro和Ala53Thr致病突变的重组pEGFP-C3-SNCA载体。获得了转染后的单克隆PC12细胞，其中三种SNCA基因亚型均有稳定表达。
