白血病细胞株KG1中Mxi1突变基因真核表达载体的构建

Construct Mxi1 Mutation Gene Expression Vector from Leukemia Cell Line KG1

目的构建Max结合蛋白1(Max interacting protein 1,Mxi1)基因的野生型和突变型真核表达载体,观察表达载体转染真核细胞后蛋白表达的情况。方法将正常人和白血病细胞株KG1细胞的Mxi1野生型/突变型基因cDNA插入到红色荧光蛋白质粒pDs-red2-N1中,构建为pDs-red2-N1/Mxi1(野生型/突变型)真核表达载体;采用脂质体方法将质粒转染Cos-7细胞;荧光显微镜观察Cos-7细胞红色荧光蛋白表达情况。流式细胞仪方法检测红色荧光蛋白中Mix1的基因表达率,以此计算转染效率。转染后48h提取细胞总RNA,进行RT-PCR检测Mxi1基因表达。结果成功构建了一种pDs-red2-N1/Mxi1(野生型/突变型)真核表达载体。荧光显微镜下观察转染后Cos-7细胞可见红色荧光蛋白的表达;流式细胞仪检测野生型和突变型的转染效率以转染后前3d较高,并可持续至第6d。质粒转染Cos-7细胞后均出现阳性Mxi1基因转录产物。RT-PCR方法可以检测到Mxi1基因在Cos-7细胞中的表达。结论 Mxi1的真核表达载体构建成功,通过脂质体的方法转染真核细胞中并在其胞浆中表达。

Objective To construct the eukaryotic expression vector for Max interacting protein 1（Mxi1）.Methods The full length cDNA of Mxi1 gene obtained from fetal lymphocyte and KG1 cells were inserted into plasmid pDs-red2-N1 respectively to generate pDs-red2-N1/Mxi1（wild/mutation type）.Then the recombinant vector was transfected into Cos-7 cells via liposome.48 hours post transfection,mRNA of Mxi1 gene was detected by RT-PCR and Mxi1 protein expression was detected by flow cytometry and fluorescence microscope in the Cos-7 cells.Results The eukaryotic expression vector of Mxi1 was constructed and transfected into eukaryotic cells successfully.The expression of red fluorescence protein in the transfected Cos-7 cells was observed under fluorescence microscope which implied the expression of Mxi1.The transfect efficiency of both wild and mutation type were in a high level in 3 days after transfected,which lasted to 6 d.RT-PCR amplified the total RNA extracted from the transfected Cos-7 cells could find increased mRNA level of Mxi1 gene.Conclusion We successfully constructed the eukaryote expression vector for Mxi1 gene;Cos-7 cells transfected with the vector via liposome could express Mxi1 protein.These could be useful for the further study of the Myc gene modulation.