人DNA聚合酶δ2真核载体的构建及在HEK293细胞中的表达

Construction of eukaryotic expression vectors of human DNA polymerase δ2 and their expression in HEK293 cells

目的构建人DNA聚合酶δ2(DNA Polδ2)真核表达载体,转染人胚胎肾细胞(HEK293)并观察其表达水平。方法用逆转录PCR(RT-PCR)方法从人胚肺成纤维细胞(WI38细胞)扩增目的基因DNA Polδ2cDNA片段,与pMD-18T载体连接,构建质粒pMD-18T-Polδ2;将目的片段DNA Polδ2从质粒pMD-18T-Polδ2切下,与真核表达载体pcDNA 3.1(+)连接,构建正义及反义真核表达载体pcDNA 3.1-Polδ2。应用脂质体转染试剂,分别将正义、反义真表达载体pcDNA 3.1-Polδ2,空载体pcDNA3.1(+)转染至HEK293细胞,同时设置阴性对照;用RT-PCR和Western blot方法检测目的基因DNA Polδ2mRNA及蛋白表达水平。结果正义和反义DNA Polδ2真核表达载体pcDNA3.1-Polδ2中插入的目的片段与GENEBANK上DNA Polδ2cDNA序列完全一致。RT-PCR方法显示正义及反义真核表达载体转染组细胞的目的基因DNA Polδ2mRNA表达均明显高于空载体转染组及阴性对照组;Western blot显示正义真核表达载体转染组细胞DNA Polδ2蛋白表达明显增强,反义真核表达载体转染组细胞DNA Polδ2蛋白表达阴性。结论人DNA Polδ2正义及反义真核表达载体pcDNA3.1-Polδ2构建成功。

Objective To construct the DNA Pol δ2 eukaryotic expression vector and to observe their transfection and expression in human embryonic kindey 293（HEK293） cells.Methods The total RNA was extracted from human embryonic lung cells（WI38 cells）,and RT-PCR was used to amplify DNA Polδ2.The PCR products were ligated to the vector of pMD-18T to construct the plasmid of pMD-18T-Polδ2.The DNA Polδ2 was cut off from the plasmid of pMD-18T,and ligated to the vector of pcDNA3.1（＋） to construct pcDNA3.1-Polδ2.The sense vector and the antisense vector of pcDNA3.1-Polδ2 and the blank vector of pcDNA3.1（＋） were transfected to HEK293 cells by the transfection reagent,while HEK293 cells were regarded as the negative control.The expression levels of mRNA of DNA Polδ2 and the level of proteins of DNA Polδ2 were detected by RT-PCR and Western blot.Results Gene sequence-measurement and BLAST analysis showed that inserted fragments of pcDNA3.1-Polδ2 were consisitent with the cDNA sequence of DNA Polδ2 published in GENEBANK.The expression of mRNA of DNA Polδ2 in the sense and the antisense vector groups were obviously higher than that in the blank vector group and the negative control group.The expression of protein of DNA Polδ2 in the sense vector group was increased significantly contrast to the blank vector group and the negative control group while that in the antisense vector group was negative.Conclusion The sense and antisense eukaryotic expression vectors of pcDNA3.1-Polδ2 are successfully constructed.