人SNCA基因真核表达载体的构建及其在PC12细胞中的表达

人SNCA基因真核表达载体的构建及其在

PC12细胞中的表达

作者：钱进军, 程言博, 刘春风, 杨亚萍, 刘康永

【摘要】 目的: 构建含人野生型及致病突变A30P、 A53T SNCA基因的重组真核表达载体pEGFP

C3

SNCA, 并通过稳定转染

synuclein(SNCA)

获得过表达人野生型及致病突变A30P、 A53T α的单克隆PC12细胞株。方法: 通过RT

PCR方法扩增SNCA基因, T

A克隆后测序。在此基础上利用单核苷酸差异引物定点诱变法相继构建含SNCA基因编码区EcoR I、 BamH I两酶切位点的同义突变及其致病突变G88C（Ala30 Pro）、 G209A（Ala53Thr）的重组真核表达载体pEGFPpEGFP

C3C3

SNCA, 并以PCR、 双酶切、 测序鉴定。以重组质粒SNCA通过脂质体转染方法转染PC12细胞; 以G418进

行筛选; 以有限稀释法进行亚克隆获得稳定过表达人野生型及致病突变A30P、 A53T α染pEGFP

synuclein的单克隆PC12细胞株, 并以稳定转

PCR、 Western

C3的PC12细胞作为对照组细胞, 通过RT

blot及荧光显微镜鉴定各单克隆PC12细胞株。结果: PCR、 酶切及测序证明重组真核表达载体pEGFP

C3

SNCA构建成功。RT

PCR、

Western blot及荧光显微镜确认目的基因序列在PC12细胞稳定过表达。结论: 成功地构建SNCA基因WT及A30P与A53T突变型的重组真核表达载体pEGFP变A30P、 A53T α

C3

SNCA; 成功获得过表达人野生型及致病突

synuclein的单克隆PC12细胞株。

【关键词】 α synuclein（SNCA） 致病突变 真核表达载体pEGFP C3 稳定转染 PC12细胞

[Abstract] AIM: To construct recombinant eukaryotic expression vectors pEGFP

C3

SNCA containing human wild

synuclein

type (WT) and pathogenic mutations A30P, A53T α

(SNCA), and to obtain monoclonal PC12 cell lines overexpressing human wild

type and pathogenic mutations A30P, A53T α

synuclein by stable transfection. METHODS: Human wild type SNCA gene was cloned by using RTthe gene was ligated with T

PCR. By T

A extension cloning,

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 sitedirected 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 wildtype, A30P or A53T αsynuclein,

respectively, and PC12 cells stably transfected with pEGFPC3 were used as control group. These monoclonal PC12 cell lines were identified with RT

PCR, Western blot and fluorescence

microscopy. RESULTS: 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 RTPCR, Western blot and fluorescence microscope, it was comfirmed that objective gene sequences in PC12 cells were stably overexpressed. CONCLUSION: 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.

[Keywords]α

synuclein（SNCA）; pathogenic mutation;

C3; stable transfection;

eukaryotic expression vector pEGFPPC12 cell

多巴胺能神经元中出现路易小体（Lewy body, LB）是帕金森病（Pakinson’s disease, PD）的特征性病理改变, SNCA基因编码的α

synuclein在病理状态下聚集形成不溶性纤维, 构成了LB的主

要成分, 而Ala30Pro、 Ala53Thr的致病突变与家族性PD相关[1, 2]。α

synuclein及其Ala30Pro、 Ala53Thr致病突变体在PD的病理机

制中可能起着十分重要的作用, 我们通过构建含绿色荧光蛋白的SNCA同义突变基因及其Ala30Pro与Ala53Thr致病突变基因的真核表达载体pEGFP

C3

SNCA质粒, 并以此转染PC12细胞, 建立稳定表

达该基因蛋白及变异蛋白的多巴胺能PC12细胞模型。

1 材料和方法

1.1 材料 克隆载体pTZ57R与真核表达载体pEGFP

C3购自

Promega公司; E.coli DH5α菌株、 培养基RPMI1640购自Invitrogen公司; PC12细胞株购自中国科学院上海细胞所; 小牛血清购自杭州四季青公司; Taq酶购自TaKaRa公司; 总RNA提取试剂为Gibco的TRIzol试剂; 反转录PCR试剂盒购自Fermanas公司; Lipofectamine2000转染试剂盒购自GbicoBRL公司; G418购自Mediatech公司; 胰蛋白酶、 兔抗人α

synuclein mAb购自Sigma

公司; HRP标记的羊抗兔IgG的二抗购自DAKO公司; ECL显色剂购自Amersham公司。