帕金森病PINK1蛋白真核表达载体pcDNA3．1-myc／PINK1的构建及其在COS-7细胞中的表达

目的构建帕金森病PINK1基因真核表达载体pcDNA3．1-mye／PINK1，检测其转染COS-7细胞后的表达。方法用PCR方法从人cDNA文库DNA中扩增PINK1全长cDNA，该基因片段两端设计了EcoR I和BamH I限制性酶切位点。将所得片段连接到T载体上测序证实。利用重组DNA技术将PINK1的cDNA构建到真核表达载体pcDNA3．1-myc—his（-）B上，酶切鉴定。通过脂质体介导的转染技术将所构建的载体导入COS-7细胞中，体外培养，于转染48h后利用RT—PCR和Western Blotting方法检测目的基因的表达情况。主要观察指标：（1）PINK1基因cDNA扩增产物检测。（2）pcDNA3．1-myc／PINK1重组体酶切鉴定。（3）Western—blotting。结果经琼脂糖凝胶电泳及测序证实，PCR获得的片段与GenBank中登记的PINK1序列完全相同；pcDNA3．1-myc／CHIP酶切片段的长度与理论大小相符；转染48h后的COS-7细胞可检测到PINK1蛋白的表达。结论PINK1蛋白真核表达载体构建成功，在COS-7细胞中可获得表达。

Construction of pcDNA3.1-myc/PINK1 and its expression in COS-7 cells

Objective To construct eukaryotic expression vector of PINK1 (pcDNA3. 1-myc/PINK1) and verify PINK1 expression in pcDNA3.1-myc/PINK1 transfected COS-7 cells. Methods The cDNA fragment encoding human PINK1 gene was amplified by PCR method from human cDNA library. After nucleotide sequencing, this cDNA fragment was inserted into an eukaryotic expression vector pcDNA3. 1-myc-his( - )B using gene cloning and DNA recombination. The recombinant was then transferred into COS-7 cells by liposome. The expression of the target molecule was assayed by western blotting. Results The sequence of DNA fragment amplified by PCR was consistent with that published in Genbank, and digestion of the recombinant plasmid with EcoR Iand BamH Iliberated DNA fragments with expected size. PINK1 was expressed and synthesized in transfected cells after 48h culture. Conclusion An eukaryotic expression plasmid containing human PINK1 gene was successfully constructed, and it can express out objective protein, which has laid a concrete foundation for future study on PINK1.