pcDNA3.1/myc-His-DJ-1^M26I重组载体构建及其对NIH3T3细胞增殖和凋亡研究

目的构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,为研究DJ-1M26I突变与细胞增殖、凋亡的关系及建立转基因动物模型奠定基础。方法采用突变试剂盒将DJ-1蛋白第26位氨基酸进行突变,分别构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组表达载体,并采用脂质体介导的方法分别将其转染入NIH3T3细胞,500μg/mL G418压力筛选稳定克隆,对2种转染细胞在DNA水平、RNA水平和蛋白质水平进行鉴定,采用MTT染色方法和AnnexinV-FITC试剂盒进行转染阳性克隆细胞的细胞活力与细胞凋亡检测。结果 pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1M26I重组质粒转染NIH3T3细胞经G418筛选后,PCR方法检测分别获得1个和3个阳性细胞克隆,RT-PCR及western blot方法进行DJ-1-His基因表达检测,结果均证明外源插入基因的表达,MTT实验结果初步证明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞增殖速率低于正常NIH3T3细胞组（P〈0.05）,转染DJ-1基因的NIH3T3阳性细胞组细胞增殖速率与正常NIH3T3细胞相比无明显差别;细胞凋亡检测表明转染DJ-1M26I基因的NIH3T3阳性细胞组细胞凋亡率高于正常NIH3T3细胞,转染DJ-1基因的NIH3T3阳性细胞组细胞凋亡率低于正常NIH3T3细胞（P〈0.05）。结论成功构建pcDNA3.1/myc-His-DJ-1和pcDNA3.1/myc-His-DJ-1 M26I重组表达载体,成功筛选出稳定表达人DJ-1及DJ-1 M26I的NIH3T3细胞。DJ-1 M26I基因突变更易导致NIH3T3细胞的凋亡。

Construction of Expression Vector pcDNA3.1/myc-His-DJ-1^M26I and Study of its Impaction on Cell Proliferation and Apoptosis in NIH3T3 Cells

Objective To construct the recombinant expression vectors of pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I,and provid a basis for further study on the relationship of DJ-1M26I mutation and cell proliferation and apoptosis and establish transgenic animal model.Methods Using site-directed mutagenesis kit to make the 26th amino acid mutated,constructed pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I recombinant expression vectors.And then transfected them into NIH3T3 cells respectively using lipofectamine.Cells were selected with G418 at the level of 500μg/ml.Stable clones were identified on the DNA,RNA and protein levels.Using MTT assay and AnnexinV-FITC kit to detect the stable cloned cells＇ viability and apoptosis level.Results NIH3T3 cells transfected with recombinant plasmid pcDNA3.1/myc-His-DJ-1 or pcDNA3.1/myc-His-DJ-1M26I screened by G418.We obtained 1 and 3 positive clones of pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I transfected cells by PCR detected.The results of RT-PCR and western blot also showed the expression of DJ-1-His in pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I transfected cells.MTT assay demonstrated the NIH3T3 positive cells transfected with DJ-1M26I had the lower proliferation rate than normal NIH3T3 cells（p0.05）.NIH3T3 positive cells carrying the DJ-1 genes showed no significant difference compared with the normal cells NIH3T3 cells.Apoptosis test indicated that the apoptosis rate of DJ-1M26I transfected cells was higher than normal NIH3T3 cells,however the apoptosis rate of the DJ-1 transfected cells was lower than normal NIH3T3 cells（p0.05）.Conclusions Recombinant expression vectors pcDNA3.1/myc-His-DJ-1 and pcDNA3.1/myc-His-DJ-1M26I have constructed successfully.The NIH3T3 cells with stable expression of DJ-1 and DJ-1M26I have been constructed successfully.DJ-1M26I mutation can result in the apoptosis of NIH3T3 cells easily.