FXYD5基因真核表达载体的构建及其功能的初步研究

Construction of eukaryotic expression vector of rat FXYD5 gene and preliminary study of its function

目的构建大鼠离子通道相关蛋白(FXYD5)基因真核表达载体,研究其对大鼠胸主动脉平滑肌细胞(CRL-1444)的体外作用。方法以含FXYD5全长cDNA的pBluescriptⅡKS(+/-)载体为模板,并用PCR方法扩增,将其连接于真核表达质粒pcDNA3.1(+),PCR、双酶切和测序鉴定后,用脂质体包裹转染大鼠胸主动脉细胞,转染重组真核表达载体为实验组(PF组),转染空质粒PCDNA3.1(+)为阴性对照组(NC组)和未做处理的为空白对照组(BC组)。Real-time PCR定量检测各组FXYD5的表达水平,并研究该基因对CRL-1444细胞膜钠钾泵(Na^+-K^+-ATPase)活性、细胞增殖能力、迁移力的影响。结果重组的真核表达载体pcDi4A3.1(+)-FXYD5构建成功,PF组FXYD5的表达水平较BC组上调17.312倍(P<0.05),PF组的细胞膜Na^+-K^+-ATPase活性高于BC组和NC组(均P<0.05),细胞增殖能力和迁移力均高于NC组(均P<0.05)。结论成功构建了真核表达载体pcDNA3.1(+)-FXYD5,并在CRL-1444细胞中使FXYD5基因的表达水平增加,且增强CRL-1444细胞膜的Na^+-K^+-ATPase活性、细胞增殖和迁移能力。

Objective To construct eukaryotic expression vector of pcDNA3.1 （＋）-FXYD5 gene and explore its effect on rat vascular smooth muscle cells（CRL-1444）. Methods The pBluescript II KS （＋/-） vector which contained full-length FXYD5 cDNA was amplified by PCR, and cloned into pcDNA3.1 （＋） plasmid. The recombinant plasmid was verified by PCR, double enzyme digestion analysis and DNA sequencing. CRL-1444 cells were transfected with pcDNA3.1 （＋） -FXYD5 using Lipofectamin 2000 as experimental group（PF）, transfected with PCDNA3.1（＋） as negative control group（NC） and not transfected as blank control group （BC）. The expression of FXYD5 was measured quantitatively by real-time PCR. The effects of the gene on membrane Na＋-K＋-ATPase activity, proliferation and migration of CRL-1444 cells were investi- gated. Results The FXYD5 gene was successfully cloned into pcDNA3.1（＋）. In comparison with BC group, the expres- sion of FXYD5 in PF group was increased by 17.312 （P〈0.05）. The membrane Na＋-K＋-ATPase activity was significantly higher in PF group than in BC group and NC group （P〈0.05）. The ability of cell proliferation and migration was significantly higher in PF group than in NC group （P〈0.05）. Conclusion The eukaryotic expression vector pcDNA3.1 （＋）-FXYD5 is successfully constructed and FXYD5 expression increases in CRL-1444 cells, which enhance the cell membrane Na＋-K＋-ATPase activity, cell proliferation and migration ability.