携带人alpha突触核蛋白真核重组质粒构建及其SKNSH细胞表达_医学论文.docVIP

携带人alpha突触核蛋白真核重组质粒构建及其SKNSH细胞表达_医学论文 携带人alpha突触核蛋白真核重组质粒构建及其SKNSH细胞表达_医学论文 作者：和青 杜婷婷 宋宁 谢俊霞 姜宏 【摘要】 目的 构建携带人alpha突触核蛋白真核重组质粒，检测其在人神经母细胞瘤SKNSH细胞中的表达及对细胞存活率的影响。方法 PCR扩增alpha突触核蛋白基因，产物纯化后与pcDNA3.1(+)载体进行连接反应, 构建pcDNA3.1(+)/alpha突触核蛋白真核重组质粒。Lipofectamine法转染人神经母细胞瘤SKNSH细胞，RTPCR方法检测SKNSH细胞中alpha突触核蛋白mRNA的表达，四甲基偶氮唑盐(MTT)法检测其对细胞存活率的影响。结果 酶切鉴定及基因测序证明人alpha突触核蛋白基因已被完整、正确地插入到pcDNA3.1(+)载体中。RTPCR 结果显示该表达载体转染SKNSH细胞后，细胞内alpha突触核蛋白mRNA的表达水平明显增高，MTT方法检测结果显示该表达载体转染并未引起细胞存活率的改变。结论 alpha突触核蛋白真核重组质粒构建成功, 并可在SKNSH细胞内表达，且对细胞的存活率不产生任何影响，从而为进一步研究alpha突触核蛋白在帕金森病发病机制中的作用奠定了基础。 【关键词】 α突触核蛋白质粒帕金森病 [ABSTRACT] Objective To construct the eukaryotic expression plasmid of human alphasynuclein gene and detect its expression in human SKNSH neuroblastoma cells and its effect on cell viability. Methods PCR was performed to amplify the entire fragment of human alphasynuclein gene, and the amplified gene was inserted into the eukaryotic expression vector pcDNA3.1 (+) to form the new construct donated pcDNA3.1(+)/alphasynuclein after purification. Liposomemediated gene transfection method was used to transfect the human SKNSH cells. RTPCR was used to detect the transient expression of alphasynuclein gene. MTT assay was used to detect the changes in cell viability. Results Recombinant contained the correct and entire nucleotide sequence of human alphasynuclein gene was found to be successfully constructed via restriction enzyme digestion and sequencing methods. Levels of alphasynuclein mRNA were obviously increased in SKNSH cells with reconstructive plasmid transfection. However, no significant changes in cell viability were observed in these transfected cells. Conclusion The eukaryotic expression plasmid of human alphasynuclein gene is successfully constructed and expressed in SKNSH cells. The cell viability is not affected. All these results establish a foundation for a further study of the role of alphasynuclein in the pathogenesy of Parkinson disease.