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目的:构建含人野生型及致病突变A30P、A53TSNCA基因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-synu-clein(SNCA)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、BamHI两酶切位点的同义突变及其致病突变G88C(Ala30Pro)、G209A(Ala53Thr)的重组真核表达载体pEGFP-C3-SNCA,并以PCR、双酶切、测序鉴定。以重组质粒pEGFP-C3-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、A53Tα-synuclein的单克隆PC12细胞株,并以稳定转染pEGFP-C3的PC12细胞作为对照组细胞,通过RT-PCR、Western 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-C3-SNCA;成功获得过表达人野生型及致病突变A30P、A53Tα-synuclein的单克隆PC12细胞株。
OBJECTIVE: To construct a recombinant eukaryotic expression vector pEGFP-C3-SNCA containing the human wild-type and pathogenic mutations A30P and A53TSNCA and to obtain human wild-type and pathogenic mutations A30P, A53Tα-synu-clein ( SNCA) monoclonal PC12 cell line. Methods: The SNCA gene was amplified by RT-PCR and sequenced after T-A cloning. On the basis of this, single-nucleotide differential primer site-directed mutagenesis was used to construct the synonymous mutations containing EcoR I and BamHI sites of SNCA gene and their pathogenic mutations G88C (Ala30Pro), G209A (Ala53Thr) Recombinant eukaryotic expression vector pEGFP-C3-SNCA, and PCR, double enzyme digestion, sequencing identification. The recombinant plasmid pEGFP-C3-SNCA was transfected into PC12 cells by lipofectamine 2000. The recombinant plasmid pEGFP-C3-SNCA was transfected into PC12 cells by G418 and subcloned by limiting dilution method to obtain a single stably overexpressing human wild type and pathogenic mutation A30P, A53Tα-synuclein PC12 cells were cloned and PC12 cells stably transfected with pEGFP-C3 were used as control cells. The monoclonal PC12 cells were identified by RT-PCR, Western blot and fluorescence microscope. Results: PCR, restriction enzyme digestion and sequencing proved that the recombinant eukaryotic expression vector pEGFP-C3-SNCA was successfully constructed. RT-PCR, Western blot and fluorescence microscope confirmed that the target gene sequence was stably overexpressed in PC12 cells. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-C3-SNCA with SNCA gene WT and A30P and A53T mutations was successfully constructed. The monoclonal PC12 cell line overexpressing human wild-type and pathogenic mutations A30P and A53Tα-synuclein was successfully constructed.